In vivo delivery of oligonucleotides

ABSTRACT

This invention provides a method for the in vivo delivery of oligonucleotides. The invention utilizes the presence of one or plurality of HES linked to an oligonucleotide to deliver a nucleic acid sequence of interest into the cytoplasm of cells and tissues of live organisms. The delivery vehicle is nontoxic to cells and organisms. Since delivery is sequence-independent and crosses membranes in a receptor-independent manner, the delivered oligonucleotide can target complementary sequences in the cytoplasm as well as in the nucleus of live cells. Sequences of bacterial or viral origin can also be targeted. The method can be used for delivery of genes coding for expression of specific proteins, antisense oligonucleotides, siRNAs, shRNAs, Dicer substrates, miRNAs, anti-miRNAs or any nucleic acid sequence in a living organism. The latter include mammals, plants, and microorganisms such as bacteria, protozoa, and viruses.

BACKGROUND OF THE INVENTION Field of the Invention

This invention pertains to the field of oligonucleotide therapeutics. Inparticular, this invention provides improved in vivo delivery foroligonucleotides including modified oligonucleotides and oligonucleotidemimics.

Over the past several decades the use of oligonucleotides as therapeuticagents has been the focus of much interest. Both blockage of thetranscription of specific genes and addition of oligonucleotidesequences coding for particular proteins have been attempted astherapies for a plethora of pathologic conditions including cancer,infectious diseases, and neurodegenerative conditions. Moreover,multiple chemical approaches have been developed to address thesynthetic, immunogenic, and biophysical properties of potentialoligonucleotide-based drugs and drug formulations. However, despite somesuccess in solution and ex vivo systems, delivery of oligonucleotidesacross biologic barriers such as cell membranes and extracellularmatrices present in live organisms as well as structural components ofinfectious agents such as cell walls has been suboptimal. Thus,accessibility to molecular targets inside cells and tissues in vivo hasbeen limiting development of the oligonucleotide therapeutics field.

In recent efforts to overcome some of the limitations of the delivery ofDNA and RNA sequences, delivery vehicles composed of lipids, sugars, andproteins conjugated to or encapsulating oligonucleotide sequences ofinterest, e.g., liposomes and lipid nanoparticles, cholesterolconjugates, and antibody conjugates, have been developed. However, noneof these formulations has enabled delivery of oligonucleotide cargoesfor the field of oligonucleotide therapeutics to reach its anticipatedrole in disease treatment. Accordingly, there is a need for improved invivo delivery systems of oligonucleotide-based therapeutics.

BRIEF SUMMARY OF THE INVENTION

The invention relates to oligonucleotide complexes containing H-typeexcitonic structures (HES) and methods of making and using thesecomplexes. The invention is based in part on the important discovery ofthe inventors that the linkage of one or a plurality of HES to single,double and multiple strand oligonucleotide sequences results in anincreased delivery of the HES-oligonucleotide sequences acrossphysiologic boundaries found in in vivo systems.

One of the toughest obstacles limiting the use of RNAi and antisenseoligonucleotides, (PNAs) and PMOs in gene expression altering therapyhas been the low uptake of these compounds by eukaryotic cells, whichwith currently available delivery methodologies is compounded by thesequestration and/or degradation of the compounds that actually do enterthe cell; the latter is predominantly via endocytosis. As will beimmediately apparent to a person of skill in the art, the surprisinglyhigh efficiency with which the non-toxic HES-oligonucleotide complexesof the invention are delivered into cells through sequence independentpassive diffusion and the discovery by the inventors that theseoligonucleotides do not co-localize with lysozomes within cells,indicate that the HES-oligonucleotide delivery vehicles of the inventionhave the ability to enter all intracellular spaces/compartments. Thus,there are essentially limitless applications in for example, research,diagnostics and therapeutics arenas. In particular embodiments, theinvention pertains to the in vivo delivery of HES-oligonucleotidecomplexes containing HES and at least one therapeutic oligonucleotidefor the treatment or prevention of a disease, disorder or condition.

Moreover, with the currently available delivery methodologies theinduction of innate antiviral defenses in mammalian cells to exogenousnucleic acid sequences have likewise significantly limited thedevelopment and use of therapeutic oligonucleotides. The inventors havediscovered that HES-oligonucleotides have low toxicity (atconcentrations greater than 10 fold the determined oligonucleotide invivo cell loading level) and in fact, have surprisingly found that thechemical linkage of HES oligonucleotides does not induce the interferonresponse in a host subject (i.e., mouse) compared to that observed withother delivery vehicles. Accordingly, in additional embodiments, theinvention encompasses a method of limiting the interferon response to anadministered exogenous nucleic acid (e.g., oligonucleotide) in a host,comprising linking 1, 2, 3 or more oligonucleotides with an HES to forman HES-oligonucleotide complex and administering the HES-oligonucleotidecomplex to a subject.

In some embodiments, an HES-oligonucleotide complex delivery vehicle isused as a diagnostic to identify and/or quantitate the presence of anucleic acid of interest in vivo. In other embodiments, anHES-oligonucleotide complex delivery vehicle is used to identify thepresence of an infectious agent in a host organism such as, a virus orbacterium in a mammalian tissue. In these embodiments the alteredfluorescence that results upon the disruption of the HES of the complexcan serve as an in vivo marker for binding of one or moreHES-oligonucleotide sequences in the complex to a nucleic acid targetsequence in a cell. Thus, in some embodiments, the complexes of theinvention have both diagnostic and therapeutic-applications. Thisapproach can also be used to quantitate the number of copies of anaberrant gene in a host in vivo.

In further embodiments, the invention provides a method for detecting analtered level of a nucleic acid biomarker for a disease or disorder invivo comprising, administering to a subject an HES-oligonucleotidecontaining an oligonucleotide that specifically hybridizes with thenucleic acid biomarker, determining the level of fluorescence in thesubject, and comparing the level of fluorescence with that obtained fora control subject that has been administered the HES-oligonucleotide,wherein an altered fluorescence compared to the control indicates thatthe subject has an altered level of the nucleic acid biomarker. Thisapproach can also be used to quantitate the number of copies of anaberrant gene of host origin in vivo

In some embodiments, the disease or disorder is: cancer, fibrosis, aproliferative disease or disorder, a neurological disease or disorder,and inflammatory disease or disorder, a disease or disorder of theimmune system, a disease or disorder of the cardiovascular system, ametabolic disease or disorder, a disease or disorder of the skeletalsystem, or a disease or disorder of the skin or eyes.

In additional embodiments, the methods of the invention are used toidentify and/or distinguish between different diseases or disorders. Themethods of the invention can likewise be used to determine among otherthings, altered nucleic acid (e.g., DNA and RNA) profiles thatdistinguish between normal and diseased (e.g., cancerous) tissue orcells, discriminate between different subtypes of diseased cells (e.g.,between different cancers and subtypes of a particular cancer), todiscriminate between mutations (e.g., oncogenic mutations) giving riseto or associated with different disease states, and to identify tissuesof origin (e.g., in a metastasized tumor).

The invention provides compositions and methods for modulating nucleicacids and protein encoded or regulated by these modulated nucleic acids.In particular embodiments, the invention provides compositions andmethods for modulating the levels, expression, processing or function ofa mRNA, small non-coding RNA (e.g., miRNA), a gene or a protein. Inparticular embodiments, the invention provides a method of delivering anoligonucleotide to a cell in vivo by administering to a subject anHES-oligonucleotide complex containing the oligonucleotide. Inparticular embodiments, the oligonucleotide is a therapeuticoligonucleotide. Moreover, in some embodiments, the oligonucleotides inthe HES-oligonucleotides of the invention are therapeuticoligonucleotides, and the destruction or significant loss of HES thatresults in an increased fluorescence when the therapeutic HESoligonucleotides specifically hybridizes with target nucleic acidsindicates that the therapeutic oligonucleotides have been delivered to,and have hybridized with the target nucleic acid. Thus, in someembodiments, the invention provides a method for monitoring and/orquantitating the delivery of a therapeutic oligonucleotide to a targetnucleic acid in vivo, comprising administering to a subject, a HESoligonucleotides containing a therapeutic oligonucleotide thatspecifically hybridizes to the target nucleic acid, and determining thelevel of fluorescence in a cell or tissue of the subject, wherein anincreased fluorescence in the cell or tissue compared to a control cellor tissue indicates that that the therapeutic oligonucleotide has beendelivered to and hybridized with the target nucleic acid.

In additional embodiments, the invention is directed to compositions fordelivering therapeutic oligonucleotides to a subject, wherein thecompositions comprise one or more H-type excitonic structures (HES)operably associated with a therapeutically effective amount of atherapeutic oligonucleotide that specifically hybridizes to a nucleicacid sequence in vivo and modulates the level of a protein encoded orregulated by the nucleic acid. In some embodiments, the therapeuticoligonucleotide is from about 8 nucleotides to about 750 nucleotides inlength. In some embodiments, the therapeutic oligonucleotide is fromabout 10 nucleotides to about 100 nucleotides in length. In someembodiments, the therapeutic oligonucleotide is single stranded. Inother embodiments, the therapeutic oligonucleotide is double stranded.In additional embodiments, the HES-oligonucleotide comprises 3 or morefluorophores capable of forming one or more HES. In further embodiments,the therapeutic oligonucleotide is a member selected from: siRNA, shRNA,miRNA, a Dicer substrate, an aptamer, a decoy and antisense. In furtherembodiments, the antisense oligonucleotide is DNA or a DNA mimic.

In some embodiments, the therapeutic oligonucleotide in anHES-oligonucleotide of the invention is an antisense oligonucleotidethat specifically hybridizes to an RNA. In farther embodiments, theantisense oligonucleotide is a substrate for RNAse H when hybridized tothe RNA. In particular embodiments, the antisense oligonucleotide is agapmer. In some embodiments, the antisense oligonucleotide contains oneor more modified internucleoside linkages selected from:phosphorothioate, phosphorodithioate, phosphoramide, 3′-methylenephosphonate, O-methylphosphoroamidiate, PNA and morpholino. Inadditional embodiments, the antisense oligonucleotide contains one ormore modified nucleobases selected from C-5 propyne and 5-methyl C. Insome embodiments, at least one nucleotide of the antisenseoligonucleotide contains a modified sugar moiety comprising amodification at the 2′-position, a PNA motif, or a morpholino motif. Infurther embodiments, at least one nucleotide of the antisenseoligonucleotide contains a modified nucleoside motif selected from:2′OME, LNA, alpha LNA, 2′-Fluoro (2′F), 2′-O(CH₂)₂OCH₃(2′-MOE) and2′-OCH₃(2′-O-methyl). In some embodiments, the modified nucleoside motifis an LNA or alpha LNA in which a methylene (—CH2-)_(n) group bridgesthe 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. Infurther embodiments, the LNA or alpha LNA contains a methyl group at the5′ position.

In additional embodiments, the therapeutic oligonucleotide in anHES-oligonucleotide of the invention is an antisense oligonucleotidethat specifically hybridizes to an RNA, but the antisenseoligonucleotide is not a substrate for RNAse H when hybridized to theRNA. In some embodiments, the antisense oligonucleotide is DNA or a DNAmimic. In some embodiments, the antisense oligonucleotide contains oneor more modified internucleoside linkages selected from:phosphorothioate, phosphorodithioate, phosphoramide, 3′-methylenephosphonate, O-methylphosphoroamidiate, PNA and morpholino. Inadditional embodiments, the antisense oligonucleotide contains one ormore modified nucleobases selected from C-5 propyne and 5-methyl C. Insome embodiments, at least one nucleotide of the antisenseoligonucleotide comprises a modified sugar moiety containing amodification at the 2′-position, a PNA motif, or a morpholino motif. Infurther embodiments, each nucleoside of the oligonucleotide comprises amodified sugar moiety containing a modification at the 2′-position, aPNA motif, or a morpholino motif. In additional embodiments, theHES-oligonucleotide comprises a modified sugar moiety containing one ormore modified nucleoside motifs selected from: 2′OME, LNA, alpha LNA,2′-Fluoro (2′F), 2′-O(CH₂)₂OCH₃(2′-MOE) and 2′-OCH₃(2′-O-methyl). Insome embodiments, the modified nucleoside motif is an LNA or alpha LNAin which a methylene (—CH2-)_(n) group bridges the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2. In further embodiments, the LNAor alpha LNA contains a methyl group at the 5′ position. In furtherembodiments, each nucleoside of the oligonucleotide comprises a modifiednucleoside motifs selected from: 2′OME, LNA, alpha LNA, 2′-Fluoro (2′F),2′-O(CH₂)₂OCH₃(2′-MOE) and 2′-OCH₃(2′-O-methyl).

In further embodiments, the therapeutic oligonucleotide in anHES-oligonucleotide of the invention is an antisense oligonucleotidecontaining a sequence that specifically hybridizes to: (a) a sequencewithin 30 nucleotides of the AUG start codon of an mRNA; (b) nucleotides1-10 of a miRNA; (c) a sequence in the 5′ untranslated region of anmRNA; (d) a sequence in the 3′ untranslated region of an mRNA; (e) anintron/exon junction of an mRNA; (f) a sequence in a precursor-miRNA(pre-miRNA) or primary-miRNA (pre-miRNA) that when bound by theoligonucleotide blocks miRNA processing; and (g) an intron/exon junctionand a region 1 to 50 nucleobases 5′ of an intron/exon junction of anRNA.

In another embodiment, the invention is directed to a composition fordelivering a therapeutic oligonucleotide to a subject, wherein thecomposition comprises one or more H-type excitonic structures (HES)operably associated with a therapeutically effective amount of atherapeutic oligonucleotide that specifically hybridizes with a nucleicacid sequence in vivo and modulates the level of a protein encoded orregulated by the nucleic acid through the induction of RNA interference(RNAi). In some embodiments, the therapeutic oligonucleotide is siRNA,shRNA or a Dicer substrate. In further embodiments, the therapeuticoligonucleotide is 18-35 nucleotides in length. In some embodiments, thetherapeutic oligonucleotide is a dicer substrate and contains 2 nucleiccomplementary nucleic acid strands that are each 18-25 nucleotides inlength and contain a 2 nucleotide 3′ overhang. In some embodiments, theoligonucleotide is dsRNA or a dsRNA mimic that is processed by Dicerenzymatic activity. In additional embodiments, the therapeuticoligonucleotide is single stranded RNA or RNA mimic capable of inducingRNA interference. In some embodiments, the therapeutic oligonucleotidecontains one or more modified internucleoside linkages selected from:phosphorothioate, phosphorodithioate, phosphoramide, 3′-methylenephosphonate, O-methylphosphoroamidiate, PNA and morpholino. Inadditional embodiments, the therapeutic oligonucleotide contains one ormore modified nucleobases selected from C-5 propyne and 5-methyl C. Insome embodiments, at least one nucleotide of the antisenseoligonucleotide contains a modified sugar moiety comprising amodification at the 2′-position, a PNA motif, or a morpholino motif. Infurther embodiments, at least one nucleotide of the therapeuticoligonucleotide comprising a modified sugar motif selected from: 2′OME,LNA, alpha LNA, 2′-Fluoro (2′F), 2′-O(CH₂)₂OCH₃(2′-MOE) and2′-OCH₃(2′-O-methyl). In some embodiments, the modified nucleoside motifis an LNA or alpha LNA in which a methylene (—CH2-)_(n) group bridgesthe 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. Infurther embodiments, the LNA or alpha LNA contains a methyl group at the5′ position.

The HES-oligonucleotide complexes of the invention provide a highlyefficient in vivo delivery of oligonucleotides into cells, essentiallyhave limitless applications in modulating target nucleic acid andprotein levels and activity The HES-oligonucleotide complexes areparticularly useful in therapeutic applications.

In some embodiments, the invention the invention provides a method ofmodulating a target nucleic acid a subject comprising administering anHES-oligonucleotide complex to the subject, wherein an oligonucleotideof the complex comprises a sequence substantially complementary to thetarget nucleic acid that specifically hybridizes to and modulates levelsof the nucleic acid or interferes with its processing or function. Insome embodiments, the target nucleic acid is RNA, in further embodimentsthe RNA is mRNA or miRNA. In further embodiments, the oligonucleotidereduces the level of a target RNA by at least 10%, at least 20%, atleast 30%, at least 40% or at least 50% in one or more cells or tissuesof the subject. In some embodiments, the target nucleic acid is a DNA.

The invention also provides compositions and methods for modulatingnucleic acids and protein encoded or regulated by these modulatednucleic acids. In particular embodiments, the invention providescompositions and methods for modulating the levels, expression,processing or function of a mRNA, small non-coding RNA (e.g., miRNA), agene or a protein.

In one embodiment, the invention provides a method of inhibiting theactivity and/or reducing the expression of a target nucleic acid in asubject, comprising administering to the subject an HES-oligonucleotidecomplex comprising an oligonucleotide which is targeted to nucleic acidscomprising or encoding the nucleic acid and which acts to reduce thelevels of the nucleic acid and/or interfere with its function in thecell. In particular embodiments, the target nucleic acid is a small-noncoding RNA, such as, a miRNA. In some embodiments, the oligonucleotidecomprises a sequence substantially complementary to the target nucleicacid.

In additional embodiments, the invention provides a method of reducingthe expression of a target RNA in a subject in need of reducingexpression of said target RNA, comprising administering to said subjectan antisense HES-oligonucleotide complex. In particular embodiments, anoligonucleotide in the complex is a substrate for RNAse H when bound tosaid target mRNA. In further embodiments, the oligonucleotide is agapmer.

In an additional embodiment, the invention provides a method ofincreasing the expression or activity of a nucleic acid in a subject,comprising administering to the subject an HES-oligonucleotide complexcontaining an oligonucleotide which comprises or encodes the nucleicacid or increases the endogenous expression, processing or function ofthe nucleic acid (e.g., by binding regulatory sequences in the geneencoding the nucleic acid) and which acts to increase the level of thenucleic acid and/or increase its function in the cell. In someembodiments, the oligonucleotide comprises a sequence substantially thesame as nucleic acids comprising or encoding the nucleic acid.

The invention also encompasses a method of treating a disease ordisorder characterized by the overexpression of a nucleic acid in asubject, comprising administering to the subject an HES-oligonucleotidecomplex containing an oligonucleotide which is targeted to a nucleicacid comprising or encoding the nucleic acid and which acts to reducethe levels of the nucleic acid and/or interfere with its function in thesubject. In further embodiments, the invention encompasses a method oftreating a disease or disorder characterized by the overexpression of aprotein in a subject, comprising administering to the subject anHES-oligonucleotide complex containing an oligonucleotide which istargeted to a nucleic acid encoding the protein or decreases theendogenous expression, processing or function of the protein in thesubject. In some embodiments, the nucleic acid is DNA, mRNA or miRNA. Inadditional embodiments the oligonucleotide is selected from a siRNA,shRNA, miRNA, an anti-miRNA, a dicer substrate, an antisenseoligonucleotide, a plasmid capable of expressing a siRNA, a miRNA, aribozyme and an antisense oligonucleotide.

In an additional embodiment, the invention also encompasses a method oftreating (e.g., alleviating) a disease or disorder characterized by theaberrant expression of a protein in a subject, comprising administeringto the subject an HES-oligonucleotide complex, containing anoligonucleotide which specifically hybridizes to the mRNA encoding theprotein and alter the splicing of the target RNA (e.g., promoting exonskipping in instances where production or overproduction of a particularsplice product is implicated in disease). In some embodiments, eachnucleoside of the oligonucleotide comprises at least one modified sugarmoiety comprising a modification at the 2′-position. In particularembodiments, the modified oligonucleotide is a 2′ OME or 2′ allyl. Inadditional embodiments, the modified oligonucleotide is LNA, alpha LNA(e.g., an LNA or alpha LNA containing a steric bulk moiety at the 5′position (e.g., a methyl group). In some embodiments the oligonucleotideis a LNA or phosphorodiamidate morpholino (PMO). In some embodiments,the oligonucleotide sequence specifically hybridizes to a sequencewithin 30 nucleotides of the AUG start codon, a sequence in the 5′ or 3′untranslated region of a target RNA, or a sequence that alters thesplicing of a target mRNA. In particular the oligonucleotidespecifically hybridizes to a sequence that alters the splicing of targetmRNA in Duchenne Muscular Dystrophy (DMD). In further embodiments, thealtered splicing results in the “skipping” of exon 51 in the resultingmRNA.

In various embodiments, the invention provides compositions for use inmodulating a target nucleic acid or protein in a cell, in vivo in asubject, or ex vivo. The HES-oligonucleotide compostions of theinvention have applications in for example, treating a disease ordisorder characterized by an overexpression, underexpression and/oraberrant expression of a nucleic acid or protein in a subject in vivo orex vivo. Uses of the compositions of the invention in treating exemplarydiseases or disorders selected from: an infectious disease, cancer, aproliferative disease or disorder, a neurological disease or disorder,and inflammatory disease or disorder, a disease or disorder of theimmune system, a disease or disorder of the cardiovascular system, ametabolic disease or disorder, a disease or disorder of the skeletalsystem, and a disease or disorder of the skin or eyes are alsoencompassed by the invention.

In additional embodiments, the invention provides a method for cellnuclear reprograming. In some embodiments, an HES-oligonucleotidescontaining one or more mimics and/or inhibitor of a miRNA or a pluralityof miRNAs are administered ex vivo into cells such as, human and mousesomatic cells to reprogram the cells to have one or more properties ofinduced pluripotent stem cells (iPSCs) or embryonic stem (ES)-likepluripotent cells. The non-toxic and highly efficientHES-oligonucleotide delivery system of the invention provides a greatlyincreased efficiency of delivery method for reprogramming cells comparedto conventional oligonucleotide delivery methods (see, e.g., U.S. Publ.Nos. 2010/0075421, US 2009/0246875, US 2009/0203141, and US2008/0293143).

Definitions

The following abbreviations are used herein:

The terms “nucleic acid” or “oligonucleotide” refer to at least twonucleotides covalently linked together. A nucleic acid/oligonucleotideof the invention is preferably single-stranded or double-stranded andgenerally contains phosphodiester bonds, although in some cases, asoutlined below, nucleic acid/oligonucleotide analogs are included thathave alternate backbones, comprising, for example, phosphoramide (see,e.g., Beaucage et al. (1993) Tetrahedron 49(10):1925) and referencestherein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977)Eur. J. Biochem. 81:579; Letsinger et al. (1986) Nucl. Acids Res.14:3587; Sawai et al. (1984) Chem. Lett. 805; Letsinger et al. (1988) J.Am. Chem. Soc. 110:4470; and Pauwels et al. (1986) Chemica Scripta26:1419, the entire contents of each of which is herein incorporated byreference in its entirety), phosphorathioate (Mag et al. (1991) NucleicAcids Res. 19:1437; and U.S. Pat. No. 5,644,048, the entire contents ofeach of which is herein incorporated by reference in its entirety),phosphorodithioate (Briu et al (1989) J. Am. Chem. Soc. 111:2321),O-methylphosphoroamidiate linkages (see, e.g., Eckstein,Oligonucleoetides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see, e.g.,Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int.Ed. Engl. 31:1008; Nielsen (1993) Nature 365:566; Carlsson et al. (1996)Nature 380:207, the entire contents of each of which is hereinincorporated by reference in its entirety). Other analog nucleicacids/oligonucleotides include those with positive backbones (see, e.g.,Dempcy et al. (1995) Proc. Natl. Acad. Sci USA 92:6097, the entirecontents of each of which is herein incorporated by reference in itsentirety); non-ionic backbones (see, e.g., U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141, and 4,469,863; Angew. (1991) Chem.Intl, Ed. English 30:423; Letsinger et al. (1988) J. Am. Chem. Soc.110:4470; Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597;Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal. (1994), Bioorganic & Medicinal Chem. Lett. 4:395; Jeffs et al.(1994) J. Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996), theentire contents of each of which is herein incorporated by reference inits entirety), and non-ribose backbones, including those described inU.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASCSymposium Series 580, Carbohydrate Modifications in Antisense Research,Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids/oligonucleotidescontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids/oligonucleotides (see, e.g., Jenkins et al.(1995), Chem. Soc. Rev. pp 169-176, the entire contents of each of whichis herein incorporated by reference in its entirety). Several nucleicacid/oligonucleotide analogs are described in Rawls, C & E News Jun. 21997 page 35, which is herein incorporated by reference in itsentirety). These modifications of the ribose-phosphate backbone may bedone for example, to facilitate the addition of additional moieties suchas labels, or to increase the stability and half-life of such moleculesin physiological environments. Nucleic acid/oligonucleotide backbones ofoligonucleotides used in the invention range from about 5 nucleotides toabout 750 nucleotides. Preferred nucleic acid/oligonucleotide backbonesused in this invention range from about 5 nucleotides to about 500nucleotides, and preferably from about 10 nucleotides to about 100nucleotides in length. As used herein, the term “about” or“approximately” when used in conjunction with a number refers to anynumber within 0.25%, 0.5%, 1%, 5% or 10% of the referenced number.

The oligonucleotides in the HES-oligonucleotide complexes of theinvention are polymeric structures of nucleoside and/or nucleotidemonomers capable of specifically hybridizing to at least a region of anucleic acid target. As indicated above, HES-oligonucleotides include,but are not limited to, compounds comprising naturally occurring bases,sugars and intersugar (backbone) linkages, non-naturally occurringmodified monomers, or portions thereof (e.g., oligonucleotide analogs ormimetics) which function similarly to their naturally occurringcounterpart, and combinations of these naturally occurring andnon-naturally occurring monomers. As used herein, the term “modified” or“modification” includes any substitution and/or any change from astarting or natural oligomeric compound, such as an oligonucleotide.Modifications to oligonucleotides encompass substitutions or changes tointernucleoside linkages, sugar moieties, or base moieties, such asthose described herein and those otherwise known in the art.

The term “antisense” as used herein, refers to an oligonucleotidesequence, written in the 5′ to 3′ direction, comprises the reversecomplement of the corresponding region of a target nucleic acid and/orthat is able to specifically hybridize to the target nucleic acid underphysiological conditions. Thus, in some embodiments, the term antisenserefers to an oligonucleotide that comprises the reverse complement ofthe corresponding region of a small noncoding RNA, untranslated mRNAand/or genomic DNA sequence. In particular embodiments, an antisenseHES-oligonucleotide in a complex of the invention, once hybridized to anucleic acid target, is able to induce or trigger a reduction in targetgene expression, target gene levels, or levels of the protein encoded bythe target nucleic acid.

“Complementary,” as used herein, refers to the capacity for pairingbetween a monomeric component of an oligonucleotide and a nucleotide ina targeted nucleic acid (e.g., DNA, mRNA, and a non-coding RNA such as,a miRNA). For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA/RNA molecule, then the oligonucleotide andDNA/RNA are considered to be complementary at that position.

In the context of this application, “hybridization” means the pairing ofan oligonucleotide with a complementary nucleic acid sequence. Suchpairing typically involves hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementarynucleoside or nucleotide bases (nucleobases) of an oligonucleotide and atarget nucleic acid sequence (e.g., wherein the oligonucleotidecomprises the reverse complementary nucleotide sequence of thecorresponding region of the target nucleic acid). In particularembodiments, an oligonucleotide specifically hybridizes to a targetnucleic acid. The terms “specifically hybridizes” and specificallyhybridizable” are used interchangeably herein to indicate a sufficientdegree of complementarity such that stable and specific binding occursbetween the oligonucleotide and the target nucleic acid (i.e., DNA orRNA). It is understood that an oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. In particular embodiments, an oligonucleotide isconsidered to be specifically hybridizable when binding of theoligonucleotide to a target nucleic acid sequence interferes with thenormal function of the target nucleic acid and results in a loss oraltered utility or expression therefom. In preferred embodiments, thereis a sufficient degree of complementarity between the oligonucleotideand target nucleic acid to avoid or minimize non-specific binding of theoligonucleotide to undesired non-target sequences under the conditionsin which specific binding is desired (e.g., under physiologicalconditions in the case of in vivo assays or therapeutic treatment, andin the case of in vitro assays, under conditions in which the assays areperformed). It is well within the level of skill of scientists in theoligonucleotide field to routinely determine when conditions are optimalfor specific hybridization to a target nucleic acid with minimalnon-specific hybridization events. Thus, in some embodiments,oligonucleotides in the complexes of the invention include 1, 2, or 3base substitutions compared to the corresponding complementary sequenceof a region of a target DNA or RNA sequence to which it specificallyhybridizes. In some embodiments, the location of a non-complementarynucleobase is at the 5′ end or 3′ end of an antisense oligonucleotide.In additional embodiments, a non-complementary nucleobase is located atan internal position in the oligonucleotide. When two or morenon-complementary nucleobases are present in an oligonucleotide, theymay be contiguous (i.e., linked), non-contiguous, or both. In someembodiments, the oligonucleotides in the complexes of the invention haveat least 85%, at least 90%, or at least 95% sequence identity to atarget region within the target nucleic acid. In other embodiments,oligonucleotides have 100% sequence identity to a polynucleotidesequence within a target nucleic acid. Percent identity is calculatedaccording to the number of bases that are identical to the correspondingnucleic acid sequence to which the oligonucleotide being compared. Thisidentity may be over the entire length of the oligomeric compound (i.e.,oligonucleotide), or in a portion of the oligonucleotide (e.g.,nucleobases 1-20 of a 27-mer may be compared to a 20-mer to determinepercent identity of the oligonucleotide to the oligonucleotide). Percentidentity between an oligonucleotide and a target nucleic acid canroutinely be determined using alignment programs and BLAST programs(basic local alignment search tools) known in the art (see, e.g.,Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

As used herein, the terms “target nucleic acid” and “nucleic acidencoding a target” are used to encompass any nucleic acid capable ofbeing targeted including, without limitation, DNA encoding a givenmolecular target (i.e., a protein or polypeptide), RNA (including miRNA,pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived fromsuch RNA. Exemplary DNA functions to be interfered with includereplication, transcription and translation. The overall effect of suchinterference with target nucleic acid function is modulation of theexpression of the target molecule. In the context of the presentinvention, “modulation” means a quantitative change, either an increase(stimulation) or a decrease (inhibition), for example in the expressionof a gene. The inhibition of gene expression through reduction in RNAlevels is a preferred form of modulation according to the presentinvention.

A “chromophore” is a group, substructure, or molecule that isresponsible for the absorbance of light. Typical chromophores each havea characteristic absorbance spectrum.

A “fluorophore” is a chromophore that absorbs light at a characteristicwavelength and then re-emits the light most typically at acharacteristic different wavelength. Fluorophores are well known tothose of skill in the art and include, but are not limited to xanthenesand xanthene derivatives, rhodamine and rhodamine derivatives, cyaninesand cyanine derivatives, coumarins and coumarin derivatives, andchelators with the lanthanide ion series. A fluorophore is distinguishedfrom a chromophore which absorbs, but does not characteristicallyre-emit light.

An “H-type excitonic structure” (HES) refers to two or more fluorophoreswhose transition dipoles are arranged in a parallel configurationresulting in a splitting of the excited singlet state; transitionsbetween a ground state and an upper excited state are considered allowedand transitions between a ground state and lower excited stateforbidden. HES formation in connection with certain fluorophores isknown in the art and the invention encompasses the attachment of thesefluorophores to oligonucleotides (e.g., diagnostic and therapeuticoligonucleotides) and the use of the resulting HES-oligonucleotidesaccording to the methods described herein. Examples of HES formingfluorophores that can be used according to the methods of the inventionare disclosed herein or otherwise known in the art and include, but arenot limited to, xanthenes and xanthene derivatives, cyanine and cyaninederivatives, coumarins and chelators with the lanthanide ion series.

The term “HES-oligonucleotide” refers to a complex of one or moreoligonucleotide strands (e.g., a single strand, double strand, triplestrand or a further plurality of strands of linear or circularoligonucleotides containing the same, complementary or distinctoligonucleotide sequences) that contain 2 or more fluorphores that forman HES. The fluorophores of the HES-oligonucleotide may be attached atthe 5′ and/or 3′ terminal backbone phosphates and/or, at another basewithin an oligonucleotide or in different oligonucleotides so long asthe collective HES-oligonucleotide contains one or more HES. Thefluorophores are optionally attached to the oligonucleotide via alinker, such as a flexible aliphatic chain.

An HES-oligonucleotide may contain 1, 2, 3, 4, or more HES.Additionally, an HES in an HES-oligonucleotide may contain 2, 3, 4 ormore of the same or different fluorophores. See, e.g., Toptygin et al.,Chem. Phys. Lett 277:430-435 (1997). In some embodiments, an HES isformed as a consequence of fluorophore aggregates betweenHES-oligonucleotides of the invention. In some embodiments, an HES isformed as a consequence of fluorophore aggregates betweenoligonucleotides of the invention that are singly labeled with afluorophore capable of forming an HES.

As used herein, the terms “pharmaceutically acceptable,” or“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a subject (e.g., a mammal such as a mouse,rat, rabbit, or a primate such as a human), without the production oftherapeutically prohibitive undesirable physiological effects such asnausea, dizziness, gastric upset and the like.

As used herein, a “pharmaceutical composition comprising an antisenseoligonucleotide” refers to a composition comprising anHES-oligonucleotide complex and a pharmaceutically acceptable diluent.By way of example, a suitable pharmaceutically acceptable diluent isphosphate-buffered saline.

A “stabilizing modification” or “stabilizing motif” means providingenhanced stability, in the presence of nucleases, relative to thatprovided by 2′-deoxynucleosides linked by phosphodiester internucleosidelinkages. Thus, such modifications provide “enhanced nuclease stability”to oligonucleotides. Stabilizing modifications include at leaststabilizing nucleosides and stabilizing internucleoside linkage groups.

The term “in vivo organism” refers to a contiguous living system capableof responding to stimuli such as reproduction, growth and development,and maintenance of homeostasis as a stable whole. Examples includemammals, plants, and microorganisms such as bacteria, protozoa, andviruses.

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to humans, non-human primates, rodents, and the like, whichis to be the recipient of a particular treatment. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa human subject.

The terms “administering” and “administration” as used herein, refer toadding a chemical such as an oligonucleotide to a subject in vivo or exvivo. Thus, administering encompasses both the addition of anHES-oligonucleotide directly to a subject and also contacting cells withHES-oligonucleotide compositions and then introducing the contactedcells into a subject. In one embodiment, cells removed from a subjectare contacted with an HES-oligonucleotide and the contacted cells arethen re-introduced to the subject.

The term “contacting” refers to adding a chemical such as anoligonucleotide to an in vivo organism such as a mammal, plant,bacterium, or virus. For mammals, common routes of contacting includeperoral (through the mouth), topical (skin), transmucosal (nasal,buccal/sublingual, vaginal, ocular and rectal), inhalation (lungs),intramuscular (muscle) and intravenous (vein). For bacteria and virusescontact may be delivery inside a cell or tissue of a host organism.

“Treating” or “treatment” includes the administration of anHES-oligonucleotide to prevent or delay the onset of the symptoms,complications, or biochemical indicia of a disease, condition, ordisorder, alleviating the symptoms or arresting or inhibiting furtherdevelopment of the disease, condition, or disorder. Treatment can beprophylactic (to prevent or delay the onset of the disease, or toprevent the manifestation of clinical or subclinical symptoms thereof)or therapeutic suppression or alleviation of symptoms after themanifestation of the disease, condition, or disorder. Treatment can bewith an HES-oligonucleotide complex containing composition alone, or incombination with 1, 2, 3 or more additional therapeutic agents.

The term “therapeutically effective amount” refers to an amount of anHES-oligonucleotide complex (“therapeutic agent”) or other drugeffective to achieve a desired therapeutic result and/or to “treat” adisease or disorder in a subject. The term “therapeutically effectiveamount” may also refer to an amount required to produce a slowing ofdisease progression, an increase in survival time, and/or an improvementin one or more indicators of disease or the progression of a disease ina subject suffering from the disease. For example, in the case ofcancer, a therapeutically effective amount an HES-oligonucleotidecomplex may: reduce angiogenesis and neovascularization; reduce thenumber of cancer cells, a therapeutically effective amount anHES-oligonucleotide complex may reduce tumor size, inhibit (i.e., slowor stop) cancer cell infiltration into peripheral organs, inhibit (i.e.,slow or stop) tumor metastasis, inhibit or slow tumor growth or tumorincidence, stimulate immune responses against cancer cells and/orrelieve one or more symptoms associated with the cancer. In the case ofan infectious disease, a therapeutically effective amount anHES-oligonucleotide complex may be associated with a reduced number ofthe infectious agent (e.g., viral load) and/or in amelioration of one ormore symptoms or conditions associated with infection caused by theinfectious agent. A “therapeutically effective amount” also may refer toan amount effective, at dosages and for periods of time necessary, toachieve a desired therapeutic result. A therapeutically effective amountof an HES-oligonucleotide complex of the invention may vary according tofactors such as, the disease state, age, sex, and weight of the subject,and the ability of the HES-oligonucleotide complex to elicit a desiredresponse in the subject. A therapeutically effective amount is also onein which any toxic or detrimental effects of the HES-oligonucleotidecomplex are outweighed by the therapeutically beneficial effects.

“Therapeutic index” means the ratio of the dose of anHES-oligonucleotide complex which produces an undesired effect to thedose which causes desired effects. In the context of the presentdisclosure, an HES-oligonucleotide complex exhibits an “improvedtherapeutic index” when activity is retained, but undesired effects arereduced or absent. For example, an HES-oligonucleotide complex having animproved therapeutic index retains the ability to inhibit miRNA activitywithout resulting in undesired effects such as immunostimulatoryactivity, or, at least, without resulting in undesired effects to adegree that would prohibit administration of the complex.

As used herein a “therapeutic oligonucleotide” refers to anoligonucleotide capable of achieving a desired therapeutic result and/orto “treat” a disease or disorder in a subject or ex vivo whenadministered at sufficient doses. Such desirable results include forexample, a slowing of disease progression, an increase in survival time,and/or an improvement in one or more indicators of disease, diseaseprogression, or disease related conditions in a subject suffering fromthe disease. Exemplary therapeutic oligonucleotides include an siRNA, anshRNA, a Dicer substrate (e.g., dsRNA), an miRNA, an anti-miRNA, anantisense, a decoy, an aptamer and a plasmid capable of expressing asiRNA, a miRNA, a ribozyme, an antisense oligonucleotide, or a proteincoding sequence. Oligonucleotides such as probes and primers that arenot able to achieve a desired therapeutic result are not consideredtherapeutic oligonucleotides for the purpose of this disclosure. Onaverage, less than 1% of mRNA is a suitable target for antisenseoligonucleotides. Numerous antisense oligonucleotides suitable forincorporation to the HES-oligonucleotides of the invention are describedherein or otherwise known in the art. Likewise, suitable therapeuticoligonucleotides can routinely be designed using guidelines, algorithmsand programs known in the art (see, e.g., Aartsma-Rus et al., Mol Ther17(3) 548-553 (2009) and Reynolds et al., Nat. Biotech. 22(3):326-330(2004), and Zhang et al., Nucleic Acids Res. 31e72 (2003), the contentsof each of which is herein incorporated by reference in its entirety).Suitable therapeutic oligonucleotides can likewise routinely be designedusing commercially available programs (e.g., MysiRNA-Designer,AsiDesigner (Bioinformatics Research Center, KRIBB), siRNA Target Finder(Ambion), Block-iT RNAi Designer (Invitrogen), Gene specific siRNAselector (The Wistar Institute), siRNA Target Finder (GeneScript),siDESIGN Center (Dharmacon), SiRNA at Whitehead, siRNA Design (IDT), D:T7 RNAi Oligo Designer (Dudek P and Picard D.), sfold-software, andRNAstructure 4.5); programs available over the internet such as, humansplicing finder software (e.g., at “.umd.be/HSF/”) and Targetfinder(available at “bioit.org.cn/ao/targetfinde r”); and commercial providers(e.g., Gene Tools, LLC). In certain instances, an HES-oligonucleotideand a therapeutic oligonucleotide may be used interchangeably hereinunless the context clearly dictates otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows fields histograms of blood cells isolated from BALB/C micethree hours after an injection of 200 microliters of buffer (PBS) or aDicer substrate. The latter contains a sequence for a gene not presentin these mice. In Panel a, cells were isolated after a single ipinjection of PBS or the Dicer substrate at a concentration of 1.5 mg/kg.In Panel b, cells were isolated after an iv injection of PBS, the Dicersubstrate at a concentration of 1.5 mg/kg, or the Dicer substrate at aconcentration of 0.75 mg/kg.

FIG. 2 shows (left column) emission spectra and (right column) hplcchromatograms of individual complementary single fluorophore-labeledstrands of RNA (top two rows) before and (bottom row) after addition toeach other. The middle column of the figure shows the fluorescenceintensity of the sense strand alone (between 0 and ca. 80 sec.) followedby quenching upon addition of the antisense strand (at ca. 80 sec.).

FIG. 3 shows the fluorescence intensity of the duplex formed between alabeled sense and labeled antisense strand of RNA as a function of timeafter addition of the recombinant Dicer enzyme.

FIG. 4 shows fluorescence intensity of single blood cells from micetransgenic for eGFP. Histogram from control cells and superimposed onthat of cells exposed to a duplex RNA targeting eGFP.

DETAILED DESCRIPTION OF THE INVENTION

Molecular targets for detection and treatment of pathologic conditionssuch as cancer, infectious diseases, and neurodegenerative disorders canbe unique DNA and RNA sequences. Studies in which binding between suchtargets and probes containing complementary sequences, a process knownas hybridization, have been carried out with high precision andspecificity; moreover, these data have provided a basis for optimism fordevelopment of treatments not currently available. However, such studieshave largely been carried out under nonphysiologic conditions, e.g., insolution or in permeabilized or fixed cells and tissues. Unfortunately,when the same probes have been tried under physiologic conditions, dueto the complementary sequences' sizes and charges combined with thepresence of permeability barriers, e.g., host cell membranes,extracellular matrices, or cell walls, accessibility to these targetshas often been considerably limited resulting in reduced effectiveness.Thus, in the past decade many resources have been directed towarddeveloping methods of delivering oligonucleotide sequences capable ofblocking gene transcription and translation in vivo.

Both biologic and chemical approaches have been used to develop deliverymethods. For example, a biologic approach has been the construction ofseveral viral vectors with promoter-expressed sequences whilechemically-based delivery vehicles have been created by conjugation ofnucleic acids with a variety of molecules including cholesterol, sugars,aptamers, and antibodies. However, the most studied chemical in vivodelivery system has utilized nanoparticles wherein nucleic acids areencapsulated in liposomes which are vesicles composed of lipid bilayers.The latter when decorated with polyethylene glycol (PEG) polymer chainsfor enhanced stability are termed SNALPs and they are sometimes furthermodified with peptide ligands on the nanoparticle surface for targetingreceptors on specific cell types.

Although some success has been achieved with the above approaches, thefollowing problems have been encountered: with viral delivery, there isa high potential for triggering immunogenicity in the host.Additionally, the risk of mutations or aberrant gene expression in thehost due to mutations in the viral sequence must be monitored. As forthe in vivo chemical delivery vehicles, unfortunately, even withenhanced modifications for specificity, delivery has been shown to belacking with respect to: (1) Specific uptake by target cells. Rather,cells of the reticuloendothelial system nonspecifically take up nucleicacid constructs, particularly nanoparticles, by a phagocytic-likeprocess. (2) Even when targeting of the desired cell is successful,internalization of the probe with or without the delivery vehicle isoften into the cells' endocytic system with the oligonucleotide endingup in lysosomes where the chemical environment, e.g., low pH, can leadto (a) destruction of the nucleic acid or (b) sequestration from thetargeted mRNA in the cytoplasm or DNA in the nucleus.

In contrast to the above described delivery vehicles, the presentinvention provides a highly efficient in vitro and in vivooligonucleotide delivery system that requires the administration oforders of magnitude of less oligonucleotide to achieve therapeuticeffect than that required using conventional delivery technologies. TheHES-oligonucleotide delivery vehicles of the invention are sequenceindependent (e.g., delivery of nucleic acids, modified nucleic acids,PNAs, morpholinos) and exploit passive diffusion to bypass cellularendoctyic systems, thereby providing access to all intracellularenvironments and increasing the delivery of oligonucleotides (e.g.,therapeutic oligonucleotides such as, siRNA, shRNA, Dicer substrates(e.g., dsRNA), miRNA, anti-miRNA, decoys, aptamers and antisense to forexample, targeted RNA in the cell cytoplasm or DNA in the nucleus. Inparticular, in preferred embodiments, the invention usesHES-oligonucleotide complexes comprising an oligonucleotide and 2 ormore fluorophores capable of forming an HES to deliver a nucleic acidsequence of interest into the cytoplasm and/or nucleus of cells andtissues of an organism in vivo. The HES-oligonucleotide delivery vehicleis nontoxic to cells and organisms. The superior sequence-independentcell membrane permeability of delivery vehicles of the inventionfacilitates the ability of oligonucleotides contained in theHES-oligonucleotide complex to cross membranes in a receptor-independentmanner and leads to increased delivery and targeting of theoligonucleotide to complementary nucleic acid sequences in the cytoplasmas well as in the nucleus of live cells. HES-oligonucleotide deliverysystems of the invention can also be used to target nucleic acidsequences of bacterial or viral origin. Moreover, theHES-oligonucleotide delivery vehicles of the invention have applicationsin the delivery of a diverse array of diagnostic and functionaloligonucleotides to cells in vivo, including but not limited to,antisense oligonucleotides, siRNAs, shRNAs, Dicer substrates, ribozymes,miRNAs, anti-miRNAs, aptamers, decoys, protein coding sequences, or anynucleic acid sequence in a living organism. Such living organismsinclude, for example, mammals, plants, and microorganisms such asbacteria, protozoa, and viruses.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but also eachmember of the group individually and all possible subgroups of the maingroup, and also the main group absent one or more of the group members.The present invention also envisages the explicit exclusion of one ormore of any of the group members in the claimed invention.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

The fluorophores in the oligonucleotide complexes of the invention canbe any fluorophores in the complex that are capable of forming an HESwith a homotypic or heterotypic cognate fluorophore(s) in the complex.In some embodiments, the HES-oligonucleotide complex comprises 2fluorophores capable of forming an H-type excitonic structure. Inadditional embodiments, the HES-oligonucleotide complex comprises 3, 4,5 or more fluorophores capable of forming an H-type excitonic structure.In further embodiments, the HES-oligonucleotide complex contains fromabout 2-20, from about 2-10, from about 2-6, or from about 2-4fluorophores capable of forming an H-type excitonic structure. Inadditional embodiments, the HES-oligonucleotide complex comprises 3, 4,5 or more fluorophores capable of forming an H-type excitonic structure.Two or more fluorophores are said to quench each other in an HES whentheir aggregate fluorescence is detectably less than the aggregatefluorescence of the fluorophores when they are separated, e.g. insolution at approximately 1 uM or less. The maximum of an HES absorbancespectrum as compared with spectra of the individual fluorophores showsthe maximum absorbance wavelength to be shifted to a shorter wavelength,i.e., a blue shift. Fluorescence intensity of H-type ExcitonicStructures or aggregates (herein “HES”) exhibits an intensity less thanthose of its components. Either a blue shift in the absorbance spectrumor a decrease in fluorescence intensity behavior of the H-type excitonicstructures or aggregates can be utilized as an indicator of a signalreporter moiety. In preferred embodiments two or more fluorophores inthe HES-oligonucleotide complex increase or quench by at least 50%,preferably by at least 70%, more preferably by at least 80%, and mostpreferably by at least 90%, 95% or even at least 99%. Examples offluorophores that can form H-type excitonic structures includexanthenes, cyanines and coumarins.

In some embodiments, the HES-oligonucleotide complex contains afluorophore selected from the group consisting of: carboxyrhodamine 110,carboxytetramethylrhodamine, carboxyrhodamine-X, diethylaminocoumarinand a carbocyanine dye. In further embodiments, the HES-oligonucleotidecomplex contains a fluorophore selected from the group consisting of:Rhodamine Green™ carboxylic acid, succinimidyl ester or hydrochloride;Rhodamine Green™ carboxylic acid, trifluoroacetamide or succinimidylester; Rhodamine Green™-X succinimidyl ester or hydrochloride; RhodolGreen™ carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester;bis-(4-carboxypiperidinyl) sulfonerhodamine or di(succinimidyl, ester);5-(and-6)-carboxynaphthofluorescein, 5-(and-6)-carboxynaphthofluoresceinsuccinimidyl ester; 5-carboxyrhodamine 6G hydrochloride;6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidylester; 6-carboxyrhodamine 6G succinimidyl ester;5-(and-6)-carboxyrhodamine 6G succinimidyl ester;5-carboxy-2′,4′,5′,7′-tetrabromosulfonefluorescein succinimidyl ester orbis-(diisopropylethyl ammonium) salt; 5-carboxytetramethylrhodamine;6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine;5-carboxytetra methylrhodamine succinimidyl ester;6-carboxytetramethylrhodamine succinimidyl ester;5-(and-6)-carboxytetramethylrhodamine succinimidyl ester;6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester;6-carboxy-X-rhodamine succinimidyl ester; 5-(and-6)-carboxy-X-rhodaminesuccinimidyl ester; 5-carboxy-X-rhodamine triethylammonium salt;Lissamine™ rhodamine B sulfonyl chloride; malachite greenisothiocyanate; Rhodamine Red™-X succinimidyl ester;6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid succinimidylester; tetramethylrhodamine-5-isothiocyanate;tetramethylrhodamine-6-isothiocyanate;tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl;Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt;Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester;X-rhodamine-5-(and-6)-isothiocyanate; and the carbocyanines.

In some embodiments, the HES-oligonucleotide complex contains ahetero-HES composed of different fluorophore. In particular embodiments,the hetero-HES contains a rhodamine or rhodamine derivative and afluorescein or a fluorescein derivative or two carbocyanines. In furtherembodiments, the hetero-HES contains a fluorescein or fluoresceinderivative selected from:6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein succinimidyl ester;5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein;5-(and-6)-carboxyfluorescein;5-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether,-alanine-carboxamide, or succinimidyl ester; 5-carboxyfluoresceinsuccinimidyl ester; 6-carboxyfluorescein succinimidyl ester,5-(and-6)-carboxyfluorescein succinimidyl ester;5-(4,6-dichlorotriazinyl)aminofluorescein; 2′,7′-difluorofluorescein;eosin-5-isothiocyanate; erythrosin-5-isothiocyanate;6-(fluorescein-5-carboxamido) hexanoic acid or succinimidyl ester;6-(fluorescein-5-(and-6)-carboxamido) hexanoic acid or succinimidylester; fluorescein-5-EX succinimidyl ester;fluorescein-5-isothiocyanate; and fluorescein-6-isothiocyanate.

Oligonucleotides

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA), deoxyribonucleic acid(DNA) or mimetics thereof. This term includes oligonucleotides composedof naturally-occurring nucleobases, sugars and covalent internucleoside(backbone) linkages (i.e., “unmodified oligonucleotide), as well asoligomeric compounds having non-naturally-occurring nucleobases, sugarsand/or internucleoside linkages and/or analogs of DNA and/or RNA whichfunction in a similar manner (i.e., nucleic acid “mimetics”). Suchmimetic oligonucleotide are often preferred over native forms because ofdesirable properties such as: enhanced affinity for nucleic acid targetand increased stability in the presence of nucleases. For example, asused herein, the term “oligonucleotide” includes morpholino (MNO)wherein one or more ribose rings of the nucleotide backbone is replacedwith a morpholine ring and phosphorodiamidate morpholino oligomers(PMOs) wherein one or more ribose ring of the nucleotide backbone isreplaced with a morpholine ring and the negatively charged intersubunitlinkages are replaced by uncharged phosphorodiamidate linkages.Likewise, the term oligonucleotide encompasses PNAs in which one or moresugar phosphate backbone of an oligonucleotide is replaced with an amidecontaining backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides. Moreover the oligonucleotides may berefers to as oligomers.

The delivery of HES-oligonucleotide vehicles of the invention aresequence independent and accordingly, the oligonucleotides contained inthe HES-oligonucleotide vehicles can be any form of nucleic acid ormimetic that is known that would be desirable to be introduced into acell.

Oligonucleotides in the HES-oligonucleotide vehicles can be in the formof single-stranded, double-stranded, circular or hairpinoligonucleotides. In some embodiments, the oligonucleotides aresingle-stranded DNA, RNA, or a nucleic acid mimetic (e.g., PMO, MNO,PNA, or oligonucleotides containing one or more modified nucleotidessuch as a 2′OME and LNA). In some embodiments, the oligonucleotides aredouble-stranded DNA, RNA, nucleic acid mimetic, DNA/nucleic acidmimetic, DNA-RNA and RNA-nucleic acid mimetic.

The inventors have surprisingly discovered that complexes containingHES-oligonucleotides such as ssDNA and dsRNA display superior sequenceindependent intracellular delivery that require the administration oforders of magnitude of less oligonucleotides that that required byconventional oligonucleotide delivery vehicles. Examples ofsingle-stranded nucleic acids contained in the complexes of theinvention include, but are not limited to, antisense, siRNA, shRNA,ribozymes, miRNA, antimiRNA, triplex-forming oligonucleotides andaptamers.

In some embodiments an oligonucleotide in an HES-oligonucleotide complexis single stranded DNA (ssDNA). In preferred embodiments, at least aportion of the ssDNA oligonucleotide specifically hybridizes with atarget RNA to form an oligonucleotide-RNA duplex. In further preferredembodiments, the oligonucleotide-RNA duplex is susceptible to an RNasecleavage mechanism (e.g., RNase H). In some embodiments, a singlestranded oligonucleotide in the complex comprises at least one modifiedbackbone linkage, at least one modified sugar, and/or at least onemodified nucleobase (e.g., as described herein). In some embodiments, asingle stranded oligonucleotide in the complex comprises at least onemodified backbone linkage, at least one modified sugar, and/or at leastone modified nucleobase (e.g., as described herein) and is capable offorming an oligonucleotide-RNA duplex that is susceptible to an RNasecleavage mechanism. In particular embodiments, the single strandedoligonucleotide is a gapmer (i.e., as described herein or otherwiseknown in the art). In additional embodiments, an oligonucleotide in theHES-oligonucleotide complex comprises at least one modified backbonelinkage, at least one modified sugar, and/or at least one modifiednucleobase that decreases the sensitivity of the oligonucleotide to anRNase cleavage mechanism (e.g., as described herein). In particularembodiments, the single stranded oligonucleotide comprises at least one2′OME, LNA, MNO or PNA motif

The inventors have also surprisingly discovered that HES-oligonucleotidecomplexes containing double stranded oligonucleotides display superiorsequence independent intracellular delivery of the double strandedoligonucleotides (also in the nanomolar and mid-micromolar range) overconventional oligonucleotide delivery vehicles. Examples ofdouble-stranded DNA oligonucleotides contained in the complexes of theinvention include, but are not limited to, dsRNAi and dicer substratesand other RNA interference reagents, and sequences corresponding tostructural genes and/or control and termination regions.

In some embodiments, the oligonucleotide is a linear double-stranded RNA(dsRNA). In preferred embodiments, the ds-RNA is susceptible to an RNasecleavage mechanism (e.g., Dicer and Drosha (an RNase III enzyme)). Inadditional embodiments, the dsRNA is able to be inserted into the RNAInduced Silencing Complex (RISC) of a cell. In further embodiments, aRNA strand of the dsRNA is able to use the RISC complex to effectcleavage of an RNA target.

In additional embodiments, the HES-oligonucleotide complex contains adouble stranded oligonucleotide in which one or both oligonucleotidescontain at least one modified backbone linkage, at least one modifiedsugar, and/or at least one modified nucleobase. In preferredembodiments, the double strand oligonucleotide is susceptible to anRNase cleavage mechanism (e.g., Dicer and Drosha (an RNase III enzyme).In additional embodiments, the double stranded oligonucleotide is ableto be inserted into the RNA Induced Silencing Complex (RISC) of a cell.In further embodiments, an oligonucleotide strand of the double strandedoligonucleotide is able to use the RISC complex to effect cleavage of anRNA target.

In further embodiments the HES-oligonucleotide complex contains a triplestranded oligonucleotide. In some embodiments the oligonucleotide is atriple-stranded DNA/RNA chimeric. In some embodiments, theoligonucleotide complex contains at least one oligonucleotide comprisingat least one modified backbone linkage, at least one modified sugarand/or at least one modified nucleobase. In particular embodiments, atleast one oligonucleotide in the complex comprises at least one 2′OME,LNA, MNO or PNA motif.

Oligonucleotides in the HES-oligonucleotide vehicles are routinelyprepared linearly but can be joined or otherwise prepared to be circularand may also include branching. Separate oligonucleotides canspecifically hybridize to form double stranded compounds that can beblunt-ended or may include overhangs on one or both termini. Inparticular embodiments, double stranded oligonucleotides (e.g., dsRNAand double stranded oligonucleotide in which at least one of theoligonucleotide strands is a nucleic acid mimetic) contained in thecomplexes of the invention are between 21-25 nucleotides in length andhave 1, 2, or 3 nucleotide overhangs at either or both ends.

Oligonucleotides in the HES-oligonucleotide complexes of the inventionmay be of various lengths, generally dependent upon the particular formof nucleic acid or mimetic and its intended use. In some embodiments,nucleic acid/oligonucleotides in the HES-oligonucleotide complexes ofthe invention range from about 5 nucleotides to about 500 nucleotides,and preferably from about 10 nucleotides to about 100 nucleotides inlength.

In some embodiments, an oligonucleotide in the HES-oligonucleotidecomplex comprises at least 8 contiguous nucleobases that arecomplementary to a target nucleic acid sequence. In various relatedembodiments, an oligonucleotide in the HES-oligonucleotide complex isfrom about 8 to about 100 monomeric subunits (used interchangeably withthe term “nucleotides” herein) or from about 8 to about 50 nucleotidesin length.

In additional embodiments an oligonucleotide in the HES-oligonucleotidecomplex ranges in length from about 8 to about 30 nucleotides, fromabout 15 to about 30 nucleotides, from about 20 to about 30 nucleotides,from about 18 to 26 nucleotides, from about 19 to 25 nucleotides, fromabout 20 to 25 or from about 21 to 25 nucleotides.

In further embodiments, an oligonucleotide in the HES-oligonucleotidecomplex is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49 or 50 subunits (nucleotides) in length.In particular embodiments, the oligonucleotides are 19, 20, 21, 22, 23,24 or 25 nucleotides in length.

In particular embodiments, the HES-oligonucleotide complex contains adouble strand of RNA oligonucleotides of between 21-25 nucleotides inlength and have 1, 2, or 3 nucleotide overhangs at either or both ends.In other embodiments, the HES-oligonucleotide complex contains a doublestrand of oligonucleotides in which at least one of the oligonucleotidestrands is a nucleic acid mimetic of between 21-25 nucleotides in lengthand the double stranded oligonucleotide has a 1, 2, or 3 nucleotideoverhang at either or both ends.

Oligonucleotides Containing Modifications

HES-oligonucleotide complexes of the invention preferably includeoligonucleotides containing one or more modified internucleosidelinkages, modified sugar moieties and/or modified nucleobases. Suchmodified oligonucleotides (i.e., mimetics) are typically preferred overnative forms because of desirable properties including for example,enhanced cellular uptake, enhanced affinity for nucleic acid target,increased stability in the presence of nucleases and/or increasedinhibitory activity.

Modified Internucleoside Linkages

The term “oligonucleotide” as used herein, refers to thoseoligonucleotides that retain a phosphorus atom in their internucleosidebackbone as well as those that do not have a phosphorus atom in theirinternucleoside backbone. In some embodiments, oligonucleotides in theHES-oligonucleotide complexes of the invention comprise one or moremodified internucleoside linkages. Modified internucleoside linkages inthe oligonucleotides of the invention may include for example, anymanner of internucleoside linkages known to provide enhanced nucleasestability to oligonucleotides relative to that provided byphosphodiester internucleoside linkages. Oligonucleotides havingmodified internucleoside linkages include internucleoside linkages thatretain a phosphorus atom as well as internucleoside linkages that do notcontain phosphorus. In some embodiments the oligonucleotides comprisemodified internucleoside linkages that alternate between modified andunmodified internucleoside linkages. In some embodiments most of theinternucleoside linkages in the oligonucleotide are modified. In furtherembodiments, every internucleoside linkage in the oligonucleotide ismodified.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphodiesters, phosphotriesters, aminoalkyl-phosphotriesters, methyland other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thiono-alkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e., a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

In preferred embodiments, the HES-oligonucleotide complexes of theinvention include at least one phosphorothioate (PS) internucleosidelinkage wherein one of the nonbridging oxygen atoms in thephosphodiester bond is replaced by sulfur. Oligonucleotides containingPS internucleoside linkage form regular Watson-Crick base pairs,activate RNase H, carry negative charges for cell delivery and displayother additional desirable pharmacokinetic properties. In someembodiments the at least one modified internucleoside linkage isphosphorothioate. In some embodiments, at least 2, 3, 4, 5, 10 or 15 ofthe internucleoside linkages contained in the oligonucleotide is aphosphorothioate linkage. In some embodiments, at least 1-10, 1-20, 1-30of the modified internucleoside linkages is a phosphorothioate linkage.In some embodiments, at least 2, 3, 4, 5, 10 or 15 of the modifiedinternucleoside linkages is a phosphorothioate linkage. In additionalembodiments, each internucleoside linkage of an oligonucleotide is aphosphorothioate internucleoside linkage.

Another suitable phosphorus-containing modified internucleoside linkageis the N3′-P5′ phosphoroamidates (NPs) in which the 3′-hydroxyl group ofthe 2′-deoxyribose ring is replaced by a 3′-amino group.Oligonucleotides containing NPs internucleoside linkages exhibit highaffinity towards complementary RNA and resistance to nucleases. Sincephosphoroamidate do not induce RNase H cleavage of the target RNA,oligonucleotides containing these internucleoside linkages haveapplications in those instances where RNA integrity needs to bemaintained, such as those instances in which the oligonucleotidesmodulation mRNA splicing. In some embodiments, at least 2, 3, 4, 5, 10or 15 of the internucleoside linkages contained in the oligonucleotideis a phosphoroamidate linkage. In some embodiments, at least 1-10, 1-20,1-30 of the modified internucleoside linkages is a phosphoroamidatelinkage. In some embodiments, at least 2, 3, 4, 5, 10 or 15 of themodified internucleoside linkages is a phosphoroamidates linkage. Inadditional embodiments, each internucleoside linkage of an antisensecompound is a phosphoroamidate internucleoside linkage.

Numerous modified internucleoside linkages and their method of synthesisare known in the art and encompassed by the modifications that may becontained in the oligonucleotides of the invention. Exemplary U.S.patents that teach the preparation of phosphorus-containinginternucleoside linkages include, but are not limited to, U.S. Pat. Nos.3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897;5,194,599; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,489,677; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,527,899; 5,536,821; 5,541,306; 5,550,111;5,563,253; 5,565,555; 5,602,240; 5,571,799; 5,587,361; 5,625,050;5,646,269; 5,663,312; 5,672,697; 5,677,439; and 5,721,218; each of whichis herein incorporated by reference in its entirety.

HES-oligonucleotide complexes containing oligonucleotides that do notinclude a phosphorus atom are also encompassed by the invention.Examples of such oligonucleotides include those containing backbonesformed by short chain alkyl or cycloalkyl internucleoside linkages,mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, orone or more short chain heteroatomic or heterocyclic internucleosidelinkages. These modified backbones include, but are not limited tooligonucleotides having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; riboacetyl backbones;alkene containing backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.Methods of making oligonucleotides containing backbones that do notinclude a phosphorous atom are known in the art and include, but are notlimited to, those methods and compositions disclosed in U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,646,269; 5,663,312; 5,633,360;5,677,437; 5,677,439; 5,792,608; and each of which is hereinincorporated by reference in its entirety.

In some embodiments, oligonucleotides of the invention contain one ormore modified backbone linkages selected from: 3′-methylene phosphonate,methylene (methylimino) (also known as MMI), morpholino, locked nucleicacid, and a peptide nucleic acid linkage. The modified backbone linkagesmay be uniform or may be alternated with other linkages, particularlyphosphodiester or phosphorothioate linkages, as long as RNAse H cleavageis not supported.

In some embodiments, the HES complexes contain oligonucleotides that arenucleic acid mimetics. The term mimetic as it is applied tooligonucleotides is intended to include oligonucleotides wherein thesugar or both the sugar and the internucleotide linkage are replacedwith alternative groups.

In some embodiments, the complexes of the invention contain anoligonucleotide having one or more morpholino linkages. The RNAse andnuclease resistant properties of morpholinos make them particularlyuseful in regulating transcription in a cell. Accordingly, in someembodiments, a complex containing a morpholino unit is used to modulategene expression. In some embodiments, morpholino unit is aphosphorodiamidate morpholino. In further embodiments, all the monomericunits of the oligonucleotide correspond to a morpholino. In furtherembodiments, all the monomeric units of the oligonucleotide correspondto a phosphorodiamidate morpholino. In particular embodiments, eachmonomeric unit of the oligonucleotide corresponds to aphosphorodiamidate morpholino (PMO). In additional embodiments a complexcontaining a morpholino oligonucleotide (e.g., PMO) is used to altermRNA splicing in a subject. In additional embodiments, a complexcontaining one or more morpholino nucleobases such as a PMO, is used asan antisense agent.

In additional embodiments, an oligonucleotide a complex of the inventionis a peptide nucleic acid (PNA). PNAs are nucleic acid mimetics in whichthe sugar phosphate backbone of an oligonucleotide is replaced with anamide containing backbone. In particular embodiments, the phosphatebackbone of an oligonucleotide is replaced with an aminoethylglycinebackbone and the nucleobases are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Numerous PNAs andmethods of making PNAs are known in the art (see, e.g., Nielsen et al.,Science, 1991, 254, 1497-150 and U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference in itsentirety. PNA containing oligonucleotides provide increased stabilityand favorable hybridization kinetics and have a higher affinity for RNAthan DNA compared to unsubstituted counterpart nucleic acids and do notactivate RNAse H mediated degradation. PNAs encompassed by the inventioninclude PNA analogues including PNAs having modified backbones withpositively charged groups and/or one or more chiral constrainedstereogenic centers at the C2(alpha), such as a D-amino acid, orC5(gamma), such as an L-amino acid (e.g., L-lysine) position of one ormore monomeric units of the oligonucleotide.

The RNAse and nuclease resistant properties of PNA oligonucleotides makethem particularly useful in regulating RNA (e.g., mRNA and miRNA) in acell via a steric block mechanism. In some embodiments,HES-oligonucleotides comprise at least one PNA oligonucleotide. In someembodiments, HES-oligonucleotides comprise at least one PNAoligonucleotide and modulate gene expression by strand invasion ofchromosomal duplex DNA. In a further embodiment, HES-oligonucleotidescontain at least one PNA oligonucleotide and alter mRNA splicing in asubject. In additional embodiments, HES-oligonucleotides comprise atleast one PNA oligonucleotide such as, a PMO, and act as an antisense.

Similarly, the RNAse and nuclease resistant properties of morpholinocontaining oligonucleotides make these oligonucleotides useful inregulating RNA (e.g., mRNA and miRNA) in a cell via a steric blockmechanism. In some embodiments, HES-oligonucleotides comprise at leastone morpholino oligonucleotide such as, a PMO, and modulate geneexpression by strand invasion of chromosomal duplex DNA. In a furtherembodiment, HES-oligonucleotides comprise at least one morpholinooligonucleotide such as, a PMO, and alter mRNA splicing in a subject. Inadditional embodiments, HES-oligonucleotides comprise at least onemorpholino oligonucleotide such as, a PMO, and act as an antisense.

Additionally, the RNAse and nuclease resistant properties of bicyclicsugar-containing nucleotides make these oligonucleotides useful inregulating RNA (e.g., mRNA and miRNA) in a cell via a steric blockmechanism. In some embodiments, complexes of the invention contain atleast one bicyclic sugar containing nucleotide. In some embodiments, thebicyclic sugar containing nucleotide is a locked nucleic acid (LNA). Infurther embodiments, the LNA has a 2′-hydroxyl group linked to the 3′ or4′ carbon atom of the sugar ring. In a further embodiment, theoligonucleotide comprises at least one locked nucleic acid (LNA) inwhich a methylene (—CH2-)_(n) group bridges the 2′ oxygen atom and the4′ carbon atom wherein n is 1 or 2. In some embodiments,HES-oligonucleotides comprise at least bicyclic sugar containingnucleotide such as an LNA, and modulate gene expression by strandinvasion of chromosomal duplex DNA. In other embodiments,HES-oligonucleotides contain at least one bicyclic sugaroligonucleotide, such as an LNA, and alter mRNA splicing in a subject.In additional embodiments, HES-oligonucleotides comprise at least onebicyclic sugar oligonucleotide, such as an LNA, and act as an antisense.

Modified Sugar Moieties

In some embodiments, oligonucleotides compounds of the inventioncomprise one or more nucleosides having one or more modified sugarmoieties which are structurally distinguishable from, yet functionallyinterchangeable with, naturally occurring or synthetic unmodifiednucleobases. In further embodiments, the oligonucleotide in theHES-oligonucleotide complex comprises a modified sugar at eachnucleoside (unit).

Examples of sugar modifications useful in the oligonucleotides of theinvention include, but are not limited to, compounds comprising a sugarsubstituent group selected from: OH; F; O-, S-, or N-alkyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl.

Representative modified sugars include carbocyclic or acyclic sugars,sugars having substituent groups at one or more of their 2′, 3′ or 4′positions, sugars having substituents in place of one or more hydrogenatoms of the sugar, and sugars having a linkage between any two otheratoms in the sugar. Examples of 2′-sugar substituent groups useful inthe oligonucleotides of the invention include, but are not limited to:OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; allyl, amino; azido;thio; O-allyl; O(CH2)2SCH3; O-, S- or N-alkynyl; or O-alkyl-O-alkyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Inparticular embodiments, the oligonucleotides contain at least one2′-sugar substituent group selected from: O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides contain at least one 2′-sugarsubstituent group selected from: a C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving pharmacokinetic properties, or agroup for improving the pharmacodynamic properties of an oligonucleotidecompound, and other substituents having similar properties.

In particular embodiments, the oligonucleotides in the complexes of theinvention comprise at least one 2′-substituted sugar having a2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, aka 2′-MOE) substituent group.

In some embodiments the oligonucleotides in the complexes of theinvention comprise at least one 2′-modified nucleoside selected from thegroup: 2′-allyl (2′-CH₂—CH—CH₂), 2′-O-allyl (2′-O—CH₂—CH—CH₂),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), and 2′-acetamido (2′-O—CH₂C(—O)NR1R1wherein each R1 is independently, H or C1-C1 alkyl.

In further embodiments, the oligonucleotides in the complexes of theinvention comprise at least one 2′-substituted sugar having: a2′-dimethylaminooxyethoxy (2′-O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE) substituent group; a 2′-dimethylaminoethoxyethoxy(2′-O—CH₂—O—CH₂—N(CH₂)₂, also known as 2′-O-dimethylaminoethoxyethyl or2′-DMAEOE) substituent group; or a 2′-O-methyl (2′-O—CH₃) substituentgroup. In further embodiments, an oligonucleotide in a complex of theinvention comprises at least one 2′-substituted sugar having a 2′-fluoro(2′-F) substituent group.

In some embodiments, oligonucleotides in the complexes of the inventioncontain at least one bicyclic sugar. In specific embodiments, theoligonucleotides have at least one locked nucleic acid (LNA) in whichthe 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugarring. In a particular embodiment, the oligonucleotides comprise at leastone locked nucleic acid (LNA) in which a methylene (—CH2-)_(n) groupbridges the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2.In another embodiment, the oligonucleotide contains at least onebicyclic modified nucleoside having a bridge between the 4′ and the 2′ribosyl ring atoms wherein the bridge is selected from selected from4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2; 4′-(CH₂)₂—O-2′ (ENA); 4′-C(CH₃)₂—O-2;4′-CH(CH₃)—O-2; 4′-CH(CH₂OCH₃)—O-2; 4′-CH₂—N(OCH₃)-2; 4′-CH₂—O—N(CH₃)-2;4′-CH₂—N(R)—O-2; 4′-CH₂—CH(CH₃)-2′ and 4′-CH₂—C(—CH₂)-2 wherein R isindependently, H, a C1-C12 alkyl, or a protecting group.Oligonucleotides in the complexes of the invention may also have atleast one of the foregoing sugar configurations and an additional motifsuch as, alpha-L-ribofuranose, beta-D-ribofuranose oralpha-L-methyleneoxy (4′-CH₂—O-2′). Further LNAs useful in of theoligonucleotides of the invention and their preparation are known in theart. See, e.g., U.S. Pat. Nos. 6,268,490, 6,670,461, 7,217,805,7,314,923, and 7,399,845; WO 98/39352 and WO 99/14226; and Singh et al.,Chem. Commun., 1998, 4, 455-456, the contents of each of which is hereinincorporated by reference in its entirety.

In some embodiments, oligonucleotides in the complexes of the inventioncomprise a chemically modified furanosyl (e.g., ribofuranose) ringmoiety. Examples of chemically modified ribofuranose rings include, butare not limited to, addition of substitutent groups (including 5′ and 2′substituent groups, and particularly the 2′ position, bridging ofnon-geminal ring atoms to form bicyclic nucleic acids (BNA), replacementof the ribosyl ring oxygen atom with S, N(R), or C(R1)(R)2 (R—H, C1-C12alkyl or a protecting group) and combinations thereof. Examples ofchemically modified sugars include 2′-F-5′-methyl substituted nucleoside(see e.g., WO 2008/101157, for other disclosed 5′,2′-bis substitutednucleosides) or replacement of the ribosyl ring oxygen atom with S withfurther substitution at the 2′-position (see e.g., US20050130923) oralternatively 5′-substitution of a BNA (WO 2007/134181 wherein LNA issubstituted with for example, a 5′-methyl or a 5′-vinyl group).

Complexes containing oligonucleotides comprising at least one nucleotidehaving a similar modification to those described above, at the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide are alsoencompassed by the invention. Representative U.S. patents that teach thepreparation of 2′-modified nucleosides contained in the oligonucleotidesof the invention include, but are not limited to, U.S. Pat. Nos.5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,700,920; and 5,792,747, each of which is herein incorporated byreference in its entirety.

In some embodiments, the oligonucleotides in the complexes of theinvention have at least one heterocyclic bicyclic nucleic acid. Forexample, in some embodiments, the oligonucleotides have at least one ENAmotif (see, e.g., WO 01/49687, the contents of which are hereinincorporated by reference in its entirety).

In additional embodiments, the oligonucleotides in the complexes of theinvention have at least one replacement of a five-membered furanose ringby a six-membered ring. In at least one embodiment, the oligonucleotideshave at least one cyclohexene nucleic acid (CeNAs). They form stableduplexes with complementary DNA or RNA and protect oligonucleotidesagainst nucleolytic degradation.

In some embodiments, the oligonucleotides in the complexes of theinvention have at least one tricyclo-DNA (tcDNA).

In particular embodiments, the oligonucleotides in the complexes of theinvention contain phosphorothioate backbones and oligonucleosides withheteroatom backbones, such as —CH2-NH—O—CH2-, —CH2-N(CH3)-O—CH2-(alsoknown as a methylene (methylimino) or MMI backbone), —CH2-O—N(CH3)-CH2-,—CH2-N(CH3)-N(CH3)—CH2- and —O—N(CH3)-CH2-CH2-, and an amide backbone(see, e.g., U.S. Pat. No. 5,602,240). In additional embodiments, theoligonucleotides in the complexes of the invention have aphosphorodiamidate backbone structure. In further embodiments, theoligonucleotides in the complexes of the invention have aphosphorodiamidate morpholino (i.e., PMO) backbone structure (see, e.g.,U.S. Pat. No. 5,034,506, the contents of which are incorporated hereinin their entirety.

Modified Nucleobases

Oligonucleotides in the complexes of the invention may also contain oneor more nucleobase modifications which are structurally distinguishablefrom, yet functionally interchangeable with, naturally occurring orsynthetic unmodified nucleobases.

The terms “unmodified” or “natural” nucleobases as used herein, includethe purine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C) and uracil (U). Modified nucleobases includesynthetic and natural nucleobases such as, for example, 5-methylcytosine(5-me-C). In some embodiments, an oligonucleotide in a complex of theinvention comprises at least one 5′ methylcytosine or a C-5 propyne. Insome embodiments, each cytosine in the oligonucleotide is amethylcytosine.

Modified nucleobases are also referred to herein as heterocyclic basemoieties and include other synthetic and natural nucleobases such asxanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl(—CC—CH3) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties contained in the oligonucleotides of theinvention may also include those in which the purine or pyrimidine baseis replaced with other heterocycles such as, 7-deazaadenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that areparticularly useful for increasing the binding affinity of theoligonucleotides of the invention include 5-substituted pp′ midines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

Additional modified nucleobases that are optionally included in theoligonucleotides of the invention, include, but are not limited to,tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one), or guanidiniumG-clamps and analogs. Representative guanidino substituent groups aredisclosed in U.S. Pat. No. 6,593,466, which is hereby incorporated byreference in its entirety. Representative acetamido substituent groupsare disclosed in U.S. Pat. No. 6,147,200, which is hereby incorporatedby reference in its entirety.

Numerous modified nucleobases encompassed by the oligonucleotidescontained in the complexes of the invention and their methods ofsynthesis are known in the art, and include, for example, the modifiednucleobases disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990; Englisch et al., Angewandte Chemie, International Edition, 1991,30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289-302; Crooke, S. T. and Lebleu, B., ed., CRCPress, 1993; and U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,681,941;5,750,692; 5,830,653; 5,763,588; 6,005,096; 6,028,183 and 6,007,992 andU.S. Appl. Publ. No. 20030158403, each of which herein incorporated byreference in its entirety.

Chimeric Oligonucleotides:

The oligonucleotides in the complexes of the invention preferablycontain one or more modified internucleoside linkages, modified sugarmoieties and/or modified nucleobases. In some embodiments,oligonucleotides are chimeric oligonucleotides (e.g., chimericoligomeric compounds). The terms “chimeric oligonucleotides” or“chimeras” are oligonucleotides that contain at least 2 chemicallydistinct regions (i.e., patterns and/or orientations of motifs ofchemically modified subunits arranged along the length of theoligonucleotide) each made up of at least one monomer unit, i.e., anucleotide or nucleoside in the case of a nucleic acid basedoligonucleotide compound. Chimeric oligonucleotides have also beenreferred to as for example, hybrids (e.g., fusions) and gapmers.Representative United States patents that teach the preparation of suchchimeric oligonucleotide structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, each of which is herein incorporated by reference in itsentirety.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. By way of example,gapmers are chimeric oligonucleotides comprising a contiguous sequenceof nucleosides that is divided into 3 regions, an central region (gap)flanked by two external regions (wings). Gapmer design typicallyincludes a central region of about 5-10 contiguous 2′-deoxynucleotideswhich serves as a substrate for RNase H is typically flanked by one ortwo regions of 2′-modified oligonucleotides that provide enhanced targetRNA binding affinity, but do not support RNAse H cleavage of the targetRNA molecule. Consequently, comparable results can often be obtainedwith shorter oligonucleotides having substrate regions when chimeras areused, compared to for example, phosphorothioate deoxyoligonucleotideshybridizing to the same target region. Other chimeric oligonucleotidesrely on regions conferring for example, altered levels of bindingaffinity over the length of an oligonucleotide for its target includingregions of modified nucleosides which exhibit either increased ordecreased affinity as compared to the other regions. So called,“MOE-gapmers” have 2′-MOE modifications in the wings, often contain fullPS backbones, and frequently include 5′MeC modifications on allcytosines.

Alternatively, for those situations in which RNAse H activity may beundesirable, such as in the modulation of RNA processing, it may bepreferable to use uniformly modified oligonucleotides, such as designsusing modified oligonucleotides that do not support RNAse H activity ateach nucleotide or nucleoside position. As used in the present inventionthe term “fully modified motif” is meant to include a contiguoussequence of sugar modified nucleosides wherein essentially eachnucleoside is modified to have the same modified sugar moiety. Suitablesugar modified nucleosides for fully modified oligonucleotides of theinvention include, but are not limited to, 2′-Fluoro (2′F),2′-O(CH₂)₂OCH₃ (2′-MOE), 2′-OCH₃ (2′-O-methyl), and bicyclic sugarmodified nucleosides. In one aspect the 3′ and 5′-terminal nucleosidesare left unmodified. In a preferred embodiment, the modified nucleosidesare either 2′-MOE, 2′-F, 2′-O-Me or a bicyclic sugar modifiednucleoside.

Oligonucleotides used in the compositions of the present invention canalso be modified to have one or more stabilizing groups. In someembodiments, the stabilizing groups are attached to one or both terminiof oligonucleotides to enhance properties such as, nuclease stability.In some embodiments, the stabilizing groups are cap structures. By “capstructure or terminal cap moiety” is meant chemical modifications, whichhave been incorporated at either terminus of oligonucleotides (see forexample WO 97/26270, which is herein incorporated by reference in itsentirety). These terminal modifications may serve to protect theoligonucleotides having terminal nucleic acid molecules from exonucleasedegradation and/or may help in the delivery and/or localization of theoligonucleotide within a cell. The oligonucleotide may contain the capat the 5′-terminus (5′-cap), the 3′-terminus (3′-cap), or both the5′-terminus and the 3′-termini. In the case of double-strandedoligonucleotides, the cap may be present at either or both termini ofeither strand. Cap structures are known in the art and include, forexample, inverted deoxy abasic caps. Further 3′ and 5′-stabilizinggroups that can be used to cap one or both ends of an oligonucleotide(e.g., antisense) compound to impart nuclease stability include thosedisclosed in WO 03/004602, which is herein incorporated by reference inits entirety.

In some embodiments, the 5′-cap of an oligonucleotide contained in anHES-oligonucleotide complex of the invention includes a structure thatis an inverted abasic residue (moiety), 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety (see e.g., WO97/26270, which is herein incorporated by reference in its entirety).

In some embodiments, the 3′-cap of an oligonucleotide contained in anHES-oligonucleotide complex of the invention includes for example a4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide;4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate;1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexylphosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate;1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modifiedbase nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide;acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide;3,5-dihydroxypentyl nucleotide, 5′,5′-inverted nucleotide moiety;5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate;1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridgingor non-bridging methylphosphonate and 5′-mercapto moieties (see also thestabilizing groups disclosed in Beaucage et al., 1993, Tetrahedron49:1925; which is herein incorporated by reference in its entirety).

In some embodiments, an oligonucleotide in a complex of the inventioncomprises one or more cationic tails. In further embodiments, theoligonucleotide is conjugated with at least 1, 2, 3, 4 or morepositively-charged amino acids such as, lysine or arginine. In specificembodiments, the oligonucleotide is a PNA and one or more lysine orarginine residues are conjugated to the C-terminal end of the molecule.In a further preferred embodiment, the oligonucleotide is a PNA andcomprises from 1 to 4 lysine and/or arginine residues are conjugated toeach PNA linkage.

In one embodiment such modified oligonucleotides are prepared bycovalently attaching conjugate groups to functional groups such ashydroxyl or amino groups. Useful conjugate groups include, but are notlimited to, intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, and groups that enhance thepharmacodynamic or pharmacokinetic properties of the oligonucleotides.Typical conjugate groups include cholesterols, carbohydrates, biotin,phenazine, folate, phenanthridine and anthraquinone. Representativeconjugate groups are disclosed in WO/1993/007883 and U.S. Pat. No.6,287,860, each of which is herein incorporated by reference in itsentirety.

Conjugate groups can be attached to various positions of anoligonucleotide directly or via an optional linking group. The termlinking group is intended to include all groups amenable to attachmentof a conjugate group to an oligomeric compound. Linking groups arebivalent groups useful for attachment of chemical functional groups,conjugate groups, reporter groups and other groups to selective sites ina parent compound such as for example an oligomeric compound. In generala bifunctional linking moiety comprises a hydrocarbyl moiety having twofunctional groups. One of the functional groups is selected to bind to aparent molecule or compound of interest and the other is selected tobind essentially any selected group such as chemical functional group ora conjugate group. In some embodiments, the linker comprises a chainstructure or an oligomer of repeating units such as ethylene glycol oramino acid units. Examples of functional groups that are routinely usedin bifunctional linking moieties include., but are not limited to,electrophiles for reacting with nucleophilic groups and nucleophiles forreacting with electrophilic groups. In some embodiments, bifunctionallinking moieties include amino, hydroxyl, carboxylic acid, thiol,unsaturations (e.g., double or triple bonds), and the like. Somenonlimiting examples of bifunctional linking moieties include8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl. Furtherrepresentative linking groups are disclosed for example in WO 94/01550and WO 94/01550.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,109,124; 5,118,802; 5,218,105; 5,414,077;5,486,603; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,512,439; 5,578,718; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928, 5,688,941 and6,114,513, and U.S. Publ. Nos. 2012/0095075; 2012/0101148; and2012/0128760, the entire contents of each of which is hereinincorporated by reference in its entirety.

In additional related embodiments, the present invention includesHES-oligonucleotide complexes and/or pharmaceutical compositionscontaining HES-oligonucleotide complexes that further comprise one ormore active agents or therapeutic agents. In one embodiment the activeagent or therapeutic agent is a nucleic acid. In various embodiments,the nucleic acid is a plasmid, an immunostimulatory oligonucleotide, asiRNA, a shRNA, a miRNA, ail anti-iniRNA, a dicer substrate, a decoy, anaptamer, an antisense oligonucleotide, or a ribozyme.

Oligonucleotide Synthesis

Oligonucleotides and phosphoramidites can be synthesized and/or modifiedby methods well established in the art. Oligomerization of modified andunmodified nucleosides is performed according to literature proceduresfor DNA-like compounds (Protocols for Oligonucleotides and Analogs, Ed.Agrawal (1993), Humana Press) and/or RNA-like compounds (Scaringe,Methods (2001), 23, 206-217. Gait et al., Applications of Chemicallysynthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36.Gallo et al., Tetrahedron 57:5707-5713 (2001),) synthesis asappropriate. (see, also, Current Protocols in Nucleic Acid Chemistry,Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is herein incorporated herein by reference in its entirety).Oligonucleotides are preferably chemically synthesized usingappropriately protected reagents and a commercially availableoligonucleotide synthesizer. Suppliers of oligonucleotide synthesisreagents useful in manufacturing the oligonucleotides of the inventioninclude, but are not limited to, Proligo (Hamburg, Germany), DharmaconResearch (Lafayette, Colo., USA), Pierce Chemical (part of PerbioScience, Rockford, Ill., USA), Glen Research (Sterling, Va., USA),ChemGenes (Ashland, Mass., USA), and Cruachem (Glasgow, UK).Alternatively, oligomers may be purchased from various oligonucleotidesynthesis companies such as, for example, Dharmacon Research Inc.,(Lafayette, Colo.), Qiagen (Germantown, Md.), Proligo and Ambion.

In certain embodiments, the preparation of oligonucleotides as disclosedherein is performed according to literature procedures for DNA:Protocols for Oligonucleotides and Analogs, Agrawal, Ed., Humana Press,1993, and/or RNA: Scaringe, Methods, 2001, 23, 206-217; Gait et al.,Applications of Chemically synthesized RNA in RNA: Protein Interactions,Smith, Ed., 1998, 1-36; Gallo et al., Tetrahedron, 2001, 57, 5707-5713.Additional methods for solid-phase synthesis may be found U.S. Pat. Nos.4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,725,677; 4,973,679; and5,132,418; and Re. 34,069.

Irrespective of the particular protocol used, the oligonucleotides usedin accordance with this invention may be conveniently and routinely madethrough the well-known technique of solid phase synthesis. Equipment forsuch synthesis is sold by several vendors including, for example, GeneForge (Redwood City, Calif.). Suitable solid phase techniques, includingautomated synthesis techniques, are described in Oligonucleotides andAnalogues, a Practical Approach, F. Eckstein, Ed., Oxford UniversityPress, New York, 1991. Any other means for such synthesis known in theart may additionally or alternatively be employed (including solutionphase synthesis).

The synthesis and preparation of the bicyclic sugar modified monomersadenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, alongwith their oligomerization, and nucleic acid recognition properties havebeen described (Koshkin et al., Tetrahedron, 54:3607-3630 (1998); WO98/39352 and WO 99/14226), the contents of each of which is hereinincorporated by reference in its entirety. Other bicyclic sugar modifiednucleoside analogs such as the 4′-CH₂—S-2′ analog have also beenprepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222).Preparation of other bicyclic sugar analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (WO 98-DK393 19980914), the contentsof each of which is herein incorporated by reference in its entirety

Techniques for linking fluorophores to oligonucleotides such as thoseused according to the methods of the invention are well known in the artand can be used or routinely modified to prepare theHES-oligonucleotides of the invention. See, e.g., Connolly et al.,Nucleic Acids Res. 13:4485-4502 (1985); Dreyer et al., Proc. Natl. Acad.Sci. 86:9752-9756 (1989); Nelson et al., Nucleic Acids Res. 17:7187-7194(1989); Sproat et al., Nucleic Acids Res. 15, 6181-6196 (1987) andZuckerman et al., Nucleic Acids Res. 15:5305-5321 (1987), the contentsof each of which is herein incorporated by reference in its entirety.Many fluorophores normally contain suitable reactive sites.Alternatively, the fluorophores may be derivatized to provide reactivesites for linkage to another molecule. Fluorophores derivatized withfunctional groups for coupling to a second molecule are commerciallyavailable from a variety of manufacturers. The derivatization may be bya simple substitution of a group on the fluorophore itself, or may be byconjugation to a linker.

Fluorophores are optionally attached to the 5′ and/or 3′ terminalbackbone phosphates and/or other bases of the oligonucleotide via alinker. Various suitable linkers are known to those of skill in the artand/or are discussed below. In some embodiments, the linker is aflexible aliphatic linker. In additional embodiments, the linker is a C1to C30 linear or branched, saturated or unsaturated hydrocarbon chain.In some embodiments, the linker is a C2 to C6 linear or branched,saturated or unsaturated hydrocarbon chain. In additional embodimentsthe hydrocarbon chain linker is substituted by one or more heteroatoms,aryls; or lower alkyls, hydroxylalkyls or alkoxys.

In some embodiments, one or more fluorophores are incorporated into anoligonucleotide during automated synthesis using one or morefluoropophore-modified nucleosides, fluorophore and sugar/base/and/orlinkage modified nucleosides, and/or deoxynucleoside phosphoramidites.

In some embodiments, one or more fluorophores are incorporated into anoligonucleotide in a post-synthesis labeling reaction. Appropriatepost-synthesis labeling reactions are known in the art and can routinelybe applied or modified to synthesize the HES-oligonucleotides of theinvention. In one embodiment, one or more fluorophores are incorporatedinto an oligonucleotide in a post-synthesis labeling reaction in whichan amine- or thiol-modified nucleotide or deoxynucleotide in thesynthesized oligonucleotide is reacted with an amine- or thiol-reactivefluorophore such as, a succinimidyl ester fluorophore.

In further embodiments, one or more of the same fluorophores areintegrated into the oligonucleotide in a single reaction that involvescontacting a reactive form of the dye with an oligonucleotide containinga desired number of reactive groups capable of reacting with thefluorpohore in a suitable buffer under conditions and for an amount oftime sufficient to accomplish the integration of the fluorphores intothe oligonucleotide. The reactive groups can routinely be incorporatedinto the oligonucleotide during synthesis using standard techniques andreagents known in the art.

Formulations:

The HES-oligonucleotide complexes are optionally admixed with a suitablepharmaceutically acceptable diluent or carrier pharmaceuticallyacceptable active or inert substance for the preparation ofpharmaceutical compositions. Thus, the invention also encompassespharmaceutical compositions that include HES-oligonucleotide complexes.Compositions and methods for the formulation of pharmaceuticalcompositions are dependent upon a number of criteria, including, but notlimited to, route of administration, extent of disease, or dose to beadministered. Such considerations are well understood by those skilledin the art.

Subject doses of the HES-oligonucleotides for mucosal or local deliverytypically range from about 0.1 ug to 50 mg per administration (e.g., inthe case of exon skipping drugs such as AVI-4658 (morpholino) whereintrial doses include the administration of the drug at 30 mg/kg and 50mg/kg wk IV) which depending on the application could be given daily,weekly, or monthly and any other amount of time there between. However,dosing may be at substantially higher or lower ranges. Determination ofappropriate dosing ranges and frequency is well within the ability ofthose skilled in the art. The administration of a given dose can becarried out both by single administration in the form of an individualdose unit or else several smaller dose units.

Pharmaceutical compositions comprising HES-oligonucleotide complexesencompass any pharmaceutically acceptable salts, esters, or salts ofsuch esters, or any other oligonucleotide which, upon administration toa subject such as a mouse, rat, rabbit or human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn tophysiologically and pharmaceutically acceptable salts (i.e., salts thatretain the desired biological activity of the parent compound and do notimpart undesired toxicological effects thereto) of HES-oligonucleotidecomplexes, prodrugs, physiologically and pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents. Suitablepharmaceutically acceptable salts include, but are not limited to (a)salts formed with cations such as sodium, potassium, ammonium,magnesium, calcium, polyamines such as spermine and spermidine, etc.;(b) acid addition salts formed with inorganic acids, for examplehydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid and the like; (c) salts formed with organic acids such as,for example, acetic acid, oxalic acid, tartaric acid, succinic acid,maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid,p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonicacid, and the like; and (d) salts formed from elemental anions such aschlorine, bromine, and iodine. Prodrugs include for example, theincorporation of additional nucleosides at one or both ends of anoligonucleotide which are cleaved by endogenous nucleases within thebody, to form the active oligonucleotide.

In some embodiments, prodrug versions of the oligonucleotides of theinvention are prepared as SATE[(S-acetyl-2-thioethyl)phosphate]derivatives according to the methodsdisclosed in WO 93/24510 and WO 94/26764.

In the context of the present invention, a pharmaceutically acceptablediluent includes phosphate-buffered saline (PBS). PBS is a diluentsuitable for use in compositions to be

Pharmaceutical compositions of the invention include, but are notlimited to, solutions and formulations. These compositions may begenerated from a variety of components that include, but are not limitedto, preformed liquids.

The pharmaceutical compositions can conveniently be presented in unitdosage form and can be prepared according to conventional techniqueswell known in the pharmaceutical industry. Such techniques include thestep of bringing into association the active ingredients with thepharmaceutical diluent(s) or carrier(s). In general the formulations areprepared by uniformly and intimately bringing into association theactive ingredients with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product.

The pharmaceutical compositions can be formulated into any of manypossible dosage forms including, but not limited to, tablets, capsules,liquid syrups, soft gels, suppositories, and enemas. The compositionscan also be formulated as suspensions in aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may additionally contain one ormore stabilizers.

As used herein, the term “dose” refers to a specified quantity of apharmaceutical agent provided in a single administration. In certainembodiments, a dose may be administered in one or more boluses, tablets,or injections. For example, in certain embodiments, where subcutaneousadministration is desired and the desired dose requires a volume noteasily accommodated by a single injection, then two or more injectionsmay be used to achieve the desired dose. In certain embodiments, a dosemay be administered in two or more injections to minimize injection sitereaction in an individual.

Administration

The present invention also includes pharmaceutical compositions andformulations which include the HES-oligonucleotide complexes of theinvention. The methods of the invention can be practiced using any modeof administration that is medically acceptable, meaning any mode thatproduces a therapeutic effect without causing clinically unacceptableadverse effects (i.e., where undesired effects are to such an extent soas to prohibit administration of the HES-oligonucleotide complex). Thepharmaceutical compositions of, the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated. Thus, foruse in therapy, an effective amount of the HES-oligonucleotide can beadministered to a subject by any mode that delivers the nucleic acid tothe desired surface, e.g., mucosal or systemic. Suitable routes ofadministration include, but are not limited, to topical oral, pulmonary,parenteral, intranasal, intratracheal, inhalation, ocular, vaginal, andrectal. Such formulations and their preparation are well known by thoseskilled in the art, as are considerations for optimal dosing routes

Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal),peroral or parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. HES-oligonucleotides with at least one 2′-O-methoxyethylmodification, including chimeric molecules or molecules which may have a2′-O-methoxyethyl modification of every nucleotide sugar, are believedto be particularly useful for oral administration. Pharmaceuticalcompositions and formulations for topical administration may includetransdermal patches, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or and the likemay be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water, capsules, sachets ortablets.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivesand pharmaceutically acceptable carriers or excipients known in the art.

In certain embodiments, parenteral administration is by infusion.Infusion can be chronic or continuous or short or intermittent. Incertain embodiments, infused pharmaceutical agents are delivered viacannulae or catheters, in certain embodiments, infused pharmaceuticalagents are delivered with a pump. In certain embodiments, the compoundsand compositions as described herein are administered parenterally. Inadditional embodiments, parenteral administration is by injection. Theinjection can be delivered with a syringe or a pump. In certainembodiments, the injection is a bolus injection. In certain embodiments,the injection is administered directly to a tissue or organ. Inadditional embodiments, the parenteral administration comprisessubcutaneous or intravenous administration.

In some embodiments, an HES-oligonucleotide complex can be administeredto a subject via an oral route of administration. The subject may be amammal, such as a mouse, a rat, a dog, a guinea pig, or a non-humanprimate. In some embodiments, the subject may be a human subject. Incertain embodiments, the subject may be in need of modulation of thelevel or expression of one or more pri-miRNAs as discussed in moredetail herein. In some embodiments, compositions for administration to asubject

In the context of the present invention, a preferred means for deliveryof an HES-oligonucleotide complex employs an infusion pump such asMedtronic SyncroMed® II pump.

The antisense oligonucleotides of the invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, a subject such as a mouse, rabbit or primate,preferably a human, suspected of having a disease or disorder which canbe treated by modulating the behavior of a cell can be treated byadministering an HES-oligonucleotide complex of the invention.

In some embodiments, the HES-oligonucleotide delivery system of theinvention is combined with one or more additional oligonucleotidedelivery systems to further facilitate HES-oligonucleotide complexdelivery into a cell and/or targeted delivery of the oligonucleotide.Marcromolecular delivery systems that can be combined with theHES-oligonucleotide delivery system include, but are not limited to theuse of dendrimers, biodegradable polymers. Additional, delivery systemsthat can be combined with the HES-oligonucleotide delivery systeminclude, but are not limited to, conjugates with amino acids, sugars, ortargeting nucleic acid motifs. In particular embodiments, anHES-oligonucleotide complex is conjugated with an aptamer, peptide, orantibody (or antibody fragment) that specifically hybridizes to acertain receptor or serum protein, which modulates the half-life of thecomplex, or which facilitates the uptake of the complex.

The HES-oligonucleotide delivery system can also be covalently attachedto cholesterol molecules.

The HES-oligonucleotide complexes of the invention may be admixed,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, receptor targetedmolecules, oral, rectal, topical or other formulations.

Exemplary Modes of Action

Antisense

In some embodiments, an oligonucleotide in an HES-oligonucleotidecomplex is an antisense oligonucleotide. The term “antisenseoligonucleotide” or simply “antisense” is meant to includeoligonucleotides corresponding to single strands of nucleic acids (e.g.,DNA, RNA and nucleic acid mimetics such as PNAs morpholinos (e.g.,PMOs), and compositions containing modified nucleosides and/orinternucleoside linkages) that bind to their cognate mRNA in the cellsof the treated subject and modulate RNA function by for example,altering the translocation of target RNA to the site of proteintranslation, translation of protein from the target RNA, alteringsplicing of the target RNA (e.g., promoting exon skipping) and alteringcatalytic activity which may be engaged in or facilitated by the targetRNA, and targeting the mRNA for degradation by endogenous RNase H. Insome embodiments, the antisense oligonucleotides alter cellular activityby hybridizing specifically with chromosomal DNA. The term antisenseoligonucleotide also encompasses antisense oligonucleotides that may notbe exactly complementary to the desired target gene. Thus, the inventioncan be utilized in instances where noxi-target specific-activities arefound with antisense, or where an antisense sequence containing one ormore mismatches with the target sequence is preferred for a particularuse. The overall effect of such interference with target nucleic acidfunction is modulation of a targeted protein of interest. In the contextof the present invention, “modulation” means either an increase(Stimulation) or a decrease (inhibition) in the expression of a gene orprotein in the amount, or levels, of a small non-coding RNA, nucleicacid target, an RNA or protein associated with a small non-coding RNA,or a downstream target of the small non-coding RNA (e.g., a mRNArepresenting a protein-coding nucleic acid that is regulated by a smallnon-cooling RNA). Inhibition is a suitable form of modulation and smallnon-coding RNA is a suitable nucleic acid target. Small non-coding RNAswhose levels can be modulated include miRNA and miRNA precursors. In thecontext of the present disclosure, “modulation of function” means analteration in the function or activity of the small non-coding RNA or analteration in the function of any cellular component with which thesmall non-coding RNA has an association or downstream effect. In oneembodiment, modulation of function is an inhibition of the activity of asmall non-coding RNA.

Antisense oligonucleotides are preferably from about 8 to about 80contiguous linked nucleosides in length. In some embodiments, theantisense oligonucleotides are from about 10 to about 50 nucleosides orfrom about 13 to about 30 nucleotides. Antisense oligonucleotides of theinvention include ribozymes, antimiRNAs, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which specifically hybridize to the targetnucleic acid and modulate its expression.

The antisense oligonucleotides in accordance with this inventioncomprise from about 15 to about 30 nucleosides in length, (i.e., from 15to 30 linked nucleosides) or alternatively, from about 17 to about 25nucleosides in length. In particular embodiments, an antisenseoligonucleotide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides in length. Inadditional embodiments, an antisense oligonucleotide is from about 10 toabout 50 nucleotides, more preferably about 15 to about 30 nucleotides.

In additional embodiments, an oligonucleotide in a complex of theinvention interferes with the transcription of a target RNA of interest.In some embodiments, the oligonucleotide interferes with transcriptionof an mRNA or miRNA of interest by strand displacement. In otherembodiments, the oligonucleotide interferes with the transcription of anmRNA by forming a stable complex with a portion of a targeted gene bystrand invasion or triplex formation (triplex forming oligonucleotides(THOs), such as those containing LNAs see, e.g., U.S. Appl. Publ. No.2012/0122104, herein incorporated by reference in its entirety). Inadditional embodiments, the HES-oligonucleotides of the inventioninterfere with the transcription of a target RNA (e.g., mRNA or miRNA)by interfering with the transcription apparatus of the cell. In someembodiments, the HES-oligonucleotides are designed to specifically binda region in the 5′ end of an mRNA or the AUG start codon (e.g., within30 nucleotides of the AUG start codon) and to reduce translation. Insome embodiments, the HES-oligonucleotides are designed to specificallyhybridize to an intron/exon junction in an RNA. In some embodiments, theHES-oligonucleotides are designed to specifically bind the 3′untranslated target sequence in an RNA (e.g., mRNA). In furtherembodiments, the HES-oligonucleotides are designed to specifically bindnucleotides 1-10 of a miRNA. In additional embodiments, theHES-oligonucleotides are designed to specifically bind a sequence in aprecursor-miRNA (pre-miRNA) or primary-miRNA (pri-miRNA) that when boundby the oligonucleotide blocks miRNA processing.

In other embodiments, the HES-oligonucleotides target sites of criticalRNA secondary structure or act as steric blockers that cause truncationof the translated polypeptide. In some embodiments, theHES-oligonucleotides (e.g., PNAs and PMOs) are designed to interferewith intron excision, by for example, binding at or near a splicejunction of the targeted mRNA. In some embodiments, theHES-oligonucleotide are designed to interfere with intron excision or toincrease the expression of an alternative splice variant.

RNase H is an endogenous enzyme that specifically cleaves the RNA moietyof an RNA:DNA duplex. In some embodiments, the antisenseoligonucleotides elicit RNase H activity when bound to a target nucleicacid. In some embodiments, the oligonucleotides are DNA or nucleic acidmimetics. HES-oligonucleotides that elicit RNase H activity haveparticular advantages in for example, harnessing endogenousribonucleases to reduces targeted RNA.

One antisense design for eliciting RNase H activity is the gapmer motifdesign in which a chimeric oligonucleotide with a central block composedof DNA, either with or without phosphorothioate modifications, andnuclease resistant 5′ and 3′ flanking blocks, usually 2′-O-methyl RNAbut a wide range of 2′ modifications have been used (see Crooke, 2004).Other gapmer designs are described herein or otherwise known in the art.

In additional embodiments, antisense oligonucleotides in the complexesof the invention are designed to avoid activation of RNase H in a cell.Oligonucleotides that do not elicit RNase H activity have particularadvantages in for example, blocking transcriptional machinery (via asteric block mechanism) and altering splicing of the target RNA. In someembodiments, the oligonucleotides are designed to interfere with and/oralter intron excision, by for example, binding at or near a splicejunction of the targeted mRNA. In additional embodiments, theoligonucleotides are designed to increase the expression of analternative splice variant of a message. In one preferred embodiment,the antisense oligonucleotide of the invention is a morpholino (e.g.,PMO). In another preferred embodiment, the antisense oligonucleotide ofthe invention is a PNA.

In particular embodiments, the antisense oligonucleotide is targeted toat least a portion of a region up to 50 nucleobases upstream of anintron/exon junction of a target mRNA. More preferably the antisenseoligonucleotide is targeted to at least a portion of a region 20-24 or30-50 nucleobases upstream of an intron/exon junction of a target mRNAand which preferably does not support RNAse H cleavage of the mRNAtarget upon binding. Preferably, the antisense compound contains atleast one modification which increases binding affinity for the RNAtarget (e.g., mRNA and miRNA) and which increases nuclease resistance ofthe antisense compound.

In one embodiment, the antisense oligonucleotide comprises at least onenucleoside having a 2′ modification of its sugar moiety. In a furtherembodiment, the antisense oligonucleotide comprises at least 2, 3, 4, 5,6, 7, 8, 9, 10, 15, or 20 nucleoside having a 2′ modification of itssugar moiety. In yet a further embodiment, every nucleoside of theantisense oligonucleotide has a 2′ modification of its sugar moiety.Preferably, the 2′ modification is 2′-fluoro, 2′-OME, 2′-methoxyethyl(2′-MOE) or a locked nucleic acid (LNA). In some embodiments, themodified nucleoside motif is an LNA or alpha LNA in which a methylene(—CH2-)_(n) group bridges the 2′ oxygen atom and the 4′ carbon atomwherein n is 1 or 2. In further embodiments, the LNA or alpha LNAcontains a methyl group at the 5′ position. In some embodiments, theoligonucleotide contains a 2′ modification and at least oneinternucleoside linkage. In particular embodiment, antisenseoligonucleotide contains at least one phosphorothioate internucleosidelinkage. In one embodiment, the internucleoside linkages of theoligonucleotide alternate between phosphodiester and phosphorothioatebackbone linkages. In another embodiment, every internucleoside linkageof the oligonucleotide is a phosphorothioate linkages.

In additional preferred embodiments, the antisense oligonucleotide inthe complexes of the invention comprises at least one 3′-methylenephosphonate, linkage, LNA, peptide nucleic acid (PNA) linkage orphosphorodiamidate morpholino linkage. In further embodiments, theantisense oligonucleotide contains at least one modified nucleobase.Preferably, the modified nucleobase is a C-5 propyne or 5-methyl C.

The antisense oligonucleotide compounds of the invention can routinelybe synthesized using techniques known in the art.

RNAi—Post Transcriptional Gene Silencing

Short double-stranded RNA molecules and short hairpin RNAs (shRNAs),i.e. fold-back stem-loop structures that give rise to siRNA can induceRNA interference (RNAi). In some embodiments, an oligonucleotide in anHES-oligonucleotide complex of the invention induces RNAi. RNAioligonucleotides in the complexes of the invention include, but are notlimited to siRNAs, shRNAs and dsRNA DROSHA and/or Dicer substrates. ThesiRNAs, shRNAs, and one or both strands of the dsRNAs preferably containone or more modified internucleoside linkages, modified sugar moietiesand/or modified nucleobases described herein or otherwise known in theart. These RNAi oligonucleotides have applications including, but notlimited to, disrupting the expression of a gene(s) or polynucleotide(s)of interest in a subject. Thus, in some embodiments, theoligonucleotides in the complexes of the invention are used tospecifically inhibit the expression of target nucleic acid. In someembodiments, double-stranded RNA-mediated suppression of gene and/ornucleic acid expression is accomplished by administering a complex ofthe invention comprising a dsRNA DROSHA substrate, dsRNA Dicersubstrate, siRNA or shRNA to a subject and/or cell. Double-strandedRNA-mediated suppression of gene and nucleic acid expression may beaccomplished according to the invention by administering dsRNA, siRNA orshRNA into a subject. SiRNA may be double-stranded RNA, or a hybridmolecule comprising both RNA and DNA, e.g., one RNA strand and one DNAstrand.

siRNAs of the invention are RNA:RNA hybrid, DNA sense:RNA antisensehybrids, RNA sense: DNA antisense hybrids, and DNA:DNA hybrid duplexesnormally 21-30 nucleotides long that can associate with a cytoplasmicmulti-protein complex known as RNAi-induced silencing complex (RISC).RISC loaded with siRNA mediates the degradation of homologous mRNAtranscripts. The invention includes the use of RNAi molecules comprisingany of these different types of double-stranded molecules. In addition,it is understood that RNAi molecules may be used and introduced to cellsin a variety of forms. Accordingly, as used herein, RNAi moleculesencompass any and all molecules capable of inducing an RNAi response incells, including, but not limited to, double-stranded polynucleotidescomprising two separate strands, i.e. a sense strand and an antisensestrand, e.g., small interfering RNA (siRNA); polynucleotides comprisinga hairpin loop of complementary sequences, which forms a double-strandedregion, e.g., shRNAi molecules, and expression vectors that express oneor more polynucleotides capable of forming a double-strandedpolynucleotide alone or in combination with another polynucleotide.

In some embodiments, dsRNA oligonucleotide contained in a complex of theinvention is double-stranded and 16-30 or 18-25 nucleotides in length.In particular embodiments, the dsRNA is 21 nucleotides in length. Incertain embodiments, the dsRNA 0-7 nucleotide 3′ overhangs or 0-4nucleotide 5′ overhangs. In particular embodiments, the dsRNA has a twonucleotide 3′ overhang. In a further embodiment, the dsRNA contains twocomplementary RNA strands of 21 nucleotides in length with twonucleotide 3′ overhangs (i.e., contains a 19 nucleotide complementaryregion between the sense and antisense strands). In certain embodiments,the overhangs are UU or dTdT 3′ overhangs.

In some embodiments, an siRNA oligonucleotide in a complex of theinvention is completely complementary to the corresponding reversecomplementary strand of a target RNA. In other embodiments, the siRNAcontains 1 or 2 substitutions, deletions or insertions compared to thecorresponding reverse complementary strand of a target RNA.

In additional embodiments, the complexes of the invention comprise anRNAi oligonucleotide that is a short hairpin RNA. shRNA is a form ofhairpin RNA containing a fold-back stem-loop structure that give rise tosiRNA and is thus, likewise capable of sequence-specifically reducingexpression of a target gene. Short hairpin RNAs are generally morestable and less susceptible to degradation in the cellular environmentthan siRNAs. The stem loop structure of ShRNAs can vary in stem length,typically from 19 to 29 nucleotides in length. In certain embodiments,the complexes of the invention comprise an shRNA having a stem that is19 to 21 or 27 to 29 nucleotides in length. In additional embodiments,the shRNA has a loop size of between 4 to 30 nucleotides in length.While complete complementarity between the portion of the stem thatspecifically hybridizes to the target mRNA (antisense strand) and themRNA is preferred, the shRNA may optionally contain mismatches betweenthe two strands of the shRNA hairpin stem. For example, in someembodiments, the shRNA includes one or several G-U pairings in thehairpin stem to stabilize hairpins during propagation in bacteria.

In one embodiment, the nucleic acid target of an RNAi oligonucleotidecontained in a complex of the invention is selected by scanning thetarget RNA (e.g., mRNA or miRNA) for the occurrence of AA dinucleotidesequences. Each AA dinucleotide sequence in combination with the 3′adjacent approximately 19 nucleotides are potential siRNA target sitesbased off of which an RNAi oligonucleotide can routinely be designed. Insome embodiments, the RNAi oligonucleotide target site is not locatedwithin the 5′ and 3′ untranslated regions (UTRs) or regions near thestart codon (e.g., within approximately 75 bases of the start codon) ofthe target RNA in order to avoid potential interference of the bindingof the siRNP endonuclease complex by proteins that bind regulatoryregions of the target RNA.

RNAi oligonucleotide targeting specific polynucleotides can be readilyprepared using or routinely modifying reagents and procedures known inthe art. Structural characteristics of effective siRNA molecules havebeen identified. Elshabir et al. (2001) Nature 411:494-498 and Elshabiret al. (2001), EMBO 20:6877-6888. Accordingly, one of skill in the artwould understand that a wide variety of different siRNA molecules may beused to target a specific gene or transcript.

Enzymatic Nucleic Acids

In some embodiments, the complexes of the invention comprise anenzymatic oligonucleotide. Two preferred features of enzymaticoligonucleotides used according to the invention are that they have aspecific substrate binding site which is complementary to one or more ofthe target gene DNA or RNA regions, and that they have nucleotidesequences within or surrounding the substrate binding site which impartan RNA cleaving activity to the oligonucleotide. In some embodiments,the enzymatic oligonucleotide is a ribozyme. Ribozymes are RNA-proteincomplexes having specific catalytic domains that possess endonucleaseactivity. Exemplary ribozyme HES-oligonucleotides of the invention areformed in a hammerhead, hairpin, a hepatitis delta virus, group I intronor RNaseP RNA (in association with an RNA guide sequence) or aNeurospora VS RNA motif.

While the enzymatic oligonucleotides in the complexes of the inventionmay contain modified nucleotides described herein or otherwise known inthe art, it is important that such modifications do not lead toconformational changes that abolish catalytic activity of the enzymaticoligonucleotide. Methods of designing, producing, testing and optimizingenzymatic oligonucleotides such as, ribozymes are known in the art andare encompassed by the invention (see, e.g., WO 91/03162; WO 92/07065;WO 93/15187; WO 93/23569; WO 94/02595, WO 94/13688; EP 92110298; andU.S. Pat. No. 5,334,711, each of which is herein incorporated byreference in its entirety).

Aptamers and Decoys

In some embodiments, the HES-oligonucleotides of the invention containan aptamer and/or a decoy. As used herein, aptamers refer to asingle-stranded nucleic acid molecule (such as DNA or RNA) that assumesa specific, sequence-dependent shape and specifically hybridizes to atarget protein with high affinity and specificity. Aptamers in thecompositions of the invention are generally fewer than 100 nucleotides,fewer than 75 nucleotides, or fewer than 50 nucleotides in length. Theterm “aptamer” as used herein, encompasses mirror-image aptamer(s)(high-affinity L-enantiomeric nucleic acids such as, L-ribose orL-2′-deoxyribose units) that confer resistance to enzymatic degradationcompared to D-oligonucleotides. Methods for making and identifyingaptamers are known in the art and can routinely be modified to identifyaptamers having desirable diagnostic and/or therapeutic properties andto incorporate these aptamers into the HES-oligonucleotides of theinvention. See, e.g., Wlotzka et al., Proc. Natl. Acad. Sci.99(13):8898-8902, 2002, which is herein incorporated by reference in itsentirety.

As used herein, the term “decoy” refers to short double-stranded nucleicacids (including single-stranded nucleic acids designed to “fold back”on themselves) that mimic a site on a nucleic acid to which a factor,such as a protein, binds. Such decoys competitively inhibit and therebydecrease the activity and/or function Gf the factor. Methods for makingand identifying decoys are known in the art and can routinely bemodified to identify decoys having desirable diagnostic and/ortherapeutic properties, and to incorporate these decoys into theHES-oligonucleotides of the invention. See, e.g., U.S. Pat. No.5,716,780 to Edwards et al, which is herein incorporated by reference inits entirety.

Small Non-Coding RNA and Antagonists (e.g., miRNAs, and Anti-miRNAs

There is a need for agents that regulate gene expression via themechanisms mediated by small non-coding RNAs. The present inventionmeets this and other needs.

As used herein, the term “small non-coding RNA” is used to encompass,without limitation, a polynucleotide molecule ranging from 17 to 29nucleotides in length. In one embodiment, a small non-coding RNA is amiRNA (also known as miRNAs, Mirs, miRs, mirs, and mature miRNAs).

MicroRNAs (miRNAs), also known as “mature” miRNA”) are small(approximately 21-24 nucleotides in length), non-coding RNA moleculesthat have been identified as key regulators of development, cellproliferation, apoptosis and differentiation. Examples of particulardevelopmental processes in which miRNAs participate include stem celldifferentiation, neurogenesis, angiogenesis, hematopoiesis, andexocytosis (reviewed by Alvarez-Garcia and Miska, Development, 2005,132, 4653-4662). miRNA have been found to be aberrantly expressed indisease states, i.e., specific miRNAs are present at higher or lowerlevels in a diseased cell or tissue as compared to healthy cell ortissue.

miRNAs are believed to originate from long endogenous primary miRNAtranscripts (also known as pri-miRNAs, pri-mirs, pri-miRs orpri-pre-miRNAs) that are often hundreds of nucleotides in length (Lee,et al., EMBO J., 2002, 21(17), 4663-4670). One mechanism by which miRNAsregulate gene expression is through binding to the 3′-untranslatedregions (3′-UTR) of specific mRNAs. miRNAs nucleotide (nt) RNA moleculesthat become incorporated into the RNA-induced silencing complex (RISC)mediate down-regulation of gene expression through translationalinhibition, transcript cleavage, or both. RISC is also implicated intranscriptional silencing in the nucleus of a wide range of eukaryotes.

The present invention provides, inter alia, compositions and methods formodulating small non-coding RNA activity, including miRNA activityassociated with disease states. Certain compositions of the inventionare particularly suited for use in in vivo methods due to their improveddelivery, potent activity and/or improved therapeutic index.

The invention provides compositions and methods for modulating smallnon-coding RNAs, including miRNA. In particular embodiments, theinvention provides compositions and methods for modulating the levels,expression, processing or function of one or a plurality of smallnon-coding RNAs, such as miRNAs. Thus, in some embodiments, theinvention encompasses compositions, such as pharmaceutical compositions,comprising an HES-oligonucleotide complex having at least oneoligonucleotide specifically hybridizable with a small noncoding RNA,such as a miRNA.

In some embodiments, an oligonucleotide in an HES-oligonucleotidecomplex of the invention specifically hybridizes with or stericallyinterferes with nucleic acid molecules comprising or encoding one ormore small non-coding RNAs, such as, miRNAs. In particular embodiments,the invention provides HES-oligonucleotide complexes and methods usefulfor modulating the levels, activity, or function of miRNAs, includingthose relying on antisense mechanisms and those that are independent ofantisense mechanisms.

As used herein, the terms “target nucleic acid,” “target RNA,” “targetRNA transcript” or “nucleic acid target” are used to encompass anynucleic acid capable of being targeted including, without limitation,RNA. In a one embodiment, the target nucleic acids are non-codingsequences including, but not limited to, miRNAs and miRNA precursors. Ina preferred embodiment, the target nucleic acid is a miRNA, which mayalso be referred to as the miRNA. An oligonucleotide is “targeted to amiRNA” when an oligonucleotide comprises a sequence substantially,including 100% complementary to a miRNA.

As used herein, oligonucleotides are “substantially complementary” tofor example, an RNA such as a small non-coding RNA, when they arecapable of specifically hybridizing to the small non-coding RNA underphysiologic conditions. In some embodiments, an oligonucleotide is“targeted to a miRNA” when an oligonucleotide comprises a sequencesubstantially, including 100% complementary to at least 8 contiguousnucleotides of a miRNA. In some embodiments, an oligonucleotide in acomplex of the invention specifically hybridizes to an miRNA and rangesin length from about 8 to about 21 nucleotides, from about 8 to about 18nucleotides, or from about 8 to about 14 nucleotides. In additionalembodiments, the oligonucleotide specifically hybridizes to an miRNA andranges in length from about 12 to about 21 nucleotides, from about 12 toabout 18 nucleotides, or from about 12 to about 14 nucleotides. Inparticular embodiments, the oligonucleotides are 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or 21 monomer subunits (nucleotides) inlength. In certain embodiments, oligonucleotides, the oligonucleotidesare 14, 15, 16, 17 or 18 monomer subunits (nucleotides) in length.

In particular embodiments, the oligonucleotide has full lengthcomplementarity to the miRNA, In other embodiments, the lengthcomplementarily between the oligonucleotide and the target nucleic acidas well as up to 3 “mismatches” between the oligonucleotide and thetarget miRNA such that the oligonucleotide is still capable ofhybridizing with the target miRNA and the function of theoligonucleotide is not substantially impaired. In other embodiments, theoligonucleotide contains a truncation or expansion with respect to thelength of target miRNA by up to 6 nucleosides, at either the 3′ or 5′end, or at both the 3′ and 5′ end of the oligonucleotide. In certainembodiments, the oligonucleotide is truncated by 1 or 2 nucleosidescompared with the length of the target miRNA. As a non-limiting example,if the target miRNA is 22 nucleotides in length, the oligonucleotidewhich has essentially full length complementarity may be 20 or 21nucleotides in length. In a particular embodiment, the oligonucleotideis truncated by 1 nucleotide on either the 3′ or 5′ end compared to themiRNA.

In some embodiments, the invention provides a method of modulating asmall non-coding RNA comprising contacting a cell with anHES-oligonucleotide complex, wherein an oligonucleotide of theHES-oligonucleotide complex comprises a sequence substantiallycomplementary to the small non-coding RNA, a small non-coding RNAprecursor (e.g., a miRNA precursor), or a nucleic acid encoding thesmall non-coding RNA. As used herein, the term “small non-coding RNAprecursor miRNA precursor” is used to encompass any longer nucleic acidsequence from which a small (mature) non-coding RNA is derived and mayinclude, without limitation, primary RNA transcripts, pri-smallnon-coding RNAs, and pre-small non-coding RNAs. For example, an “miRNAprecursor” encompasses any longer nucleic acid sequence from which amiRNA is derived and may include, without limitation, primary RNAtranscripts, pri-miRNAs, and pre-miRNAs.

The invention provides, inter alia, compositions such as pharmaceuticalcompositions, containing an HES-oligonucleotide complex containing anoligonucleotide which is targeted to nucleic acids comprising orencoding small a non-coding RNA, and which acts to modulate the levelsof the small non-coding RNA, or modulate its function. In furtherembodiments, the invention provides, a composition such as apharmaceutical composition, containing an HES-oligonucleotide complexcomprising an oligonucleotide which is targeted to a miRNA and whichacts to modulate the levels of the miRNA, or interfere with itsprocessing or function.

In some embodiments, the HES-oligonucleotide complex contains anoligonucleotide that specifically hybridizes to nucleotides 1-10 of amiRNA (i.e., the seed region). In additional embodiments, theoligonucleotide specifically hybridizes to a sequence in aprecursor-miRNA (pre-miRNA) or primary-miRNA (pri-miRNA) that when boundby the oligonucleotide blocks miRNA processing.

In some embodiments, the composition contains an HES-oligonucleotidecomplex contains an oligonucleotide which is targeted to nucleic acidscomprising or encoding a small non-coding RNA and which acts to reducethe levels of the small non-coding RNA and/or interfere with itsfunction in a cell.

In other embodiments, the composition contains an HES-oligonucleotidecomplex contains an oligonucleotide which comprises or encodes the smallnon-coding RNA or increases the endogenous expression, processing orfunction of the small non-coding RNA (e.g., by binding regulatorysequences in the gene encoding the non-coding RNA) and which acts toincrease the level of the small non-coding RNA and/or increase itsfunction in a cell.

Oligonucleotides contained in the HES-oligonucleotides of the inventioncan modulate the levels, expression or function of small non-coding RNAsby hybridizing to a nucleic acid comprising or encoding a smallnon-coding RNA nucleic acid target resulting in alteration of normalfunction. For example, non-limiting mechanisms by which theoligonucleotides might decrease the activity (including levels,expression or function) of a small non-coding RNA include facilitatingthe destruction of the small non-coding RNA through cleavage,sequestration, steric occlusion and by hybridizing to the smallnon-coding RNA and preventing it from hybridizing to, and regulating theactivity of, its normal cellular target(s).

In an additional embodiment, the invention provides a method ofinhibiting the activity of a small non-coding RNA, comprising contactinga cell with an HES-oligonucleotide complex comprising an oligonucleotidewhich is targeted to nucleic acids comprising or encoding a smallnon-coding RNA and which acts to reduce the levels of the smallnon-coding RNA and/or interfere with its function in the cell. In someembodiments, the oligonucleotide comprises a sequence substantiallycomplementary nucleic acids comprising or encoding the non-coding RNA.In particular embodiments, the small non-coding RNA is a miRNA.

In another embodiment, the invention provides a method of inhibiting theactivity of a small non-coding RNA, comprising administering to asubject an HES-oligonucleotide complex containing an oligonucleotidewhich is targeted to nucleic acids comprising or encoding a smallnon-coding RNA and which acts to reduce the levels of the smallnon-coding RNA and/or interfere with its function in the subject. Insome embodiments, the oligonucleotide comprises a sequence substantiallycomplementary nucleic acids comprising or encoding the non-coding RNA.In particular embodiments, the small non-coding RNA is a miRNA.

In an additional embodiment, the invention provides a method ofincreasing the activity of a small non-coding RNA, comprising contactinga cell with an HES-oligonucleotide complex containing an oligonucleotidewhich comprises or encodes the small non-coding RNA or increases theendogenous expression, processing or function of the small non-codingRNA (e.g., by binding regulatory sequences in the gene encoding thenon-coding RNA) and which acts to increase the level of the smallnon-coding RNA and/or increase its function in the cell. In someembodiments, the oligonucleotide comprises a sequence substantially thesame as nucleic acids comprising or encoding the non-coding RNA. In someembodiments, the oligonucleotide shares 100% identity with at least 15contiguous nucleotides, at least 20 contiguous nucleotides or over thefull-length of the small non-coding RNA sequence. In particularembodiments, the small non-coding RNA is a miRNA.

In another embodiment, the invention provides a method of increasing theactivity of a small non-coding RNA, comprising administering to asubject an HES-oligonucleotide complex containing an oligonucleotidewhich comprises or encodes the small non-coding RNA or increases theendogenous expression, processing or function of the small non-codingRNA, and which acts to increase the level of the small non-coding RNAand/or increase its function in the subject. In some embodiments, theoligonucleotide comprises a sequence substantially the same as nucleicacids comprising or encoding the non-coding RNA. In some embodiments,the oligonucleotide shares 100% identity with at least 15 contiguousnucleotides, at least 20 contiguous nucleotides or over the tall-lengthof the small non-coding RNA sequence. In particular embodiments, thesmall non-coding RNA is a miRNA.

In additional embodiments, the HES-oligonucleotide comprises a sequencesubstantially the same as nucleic acids comprising or encoding the smallnon-coding RNA. In some embodiments, the HES-oligonucleotide is a miRNAmimic. In some embodiments the miRNA mimic is double stranded. Infurther embodiments, the HES-oligonucleotide contains an miRNA mimicthat is double stranded and contains oligonucleotides of 18-23 units inlength and is blunt ended or comprises one or more 3′ overhangs of 1, 2,or 3 nucleotides. In additional embodiments, the HES-oligonucleotidecontains a single stranded miRNA mimic that is 18-23 units in length.HES-oligonucleotides containing expression vectors that express thesemiRNA mimics are also encompassed by the invention. In some embodiments,the oligonucleotide shares 100% identity with at least 15 contiguousnucleotides, at least 20 contiguous nucleotides or over the full-lengthof the small non-coding RNA sequence. In particular embodiments, thesmall non-coding RNA is a miRNA.

The invention also encompasses a method of treating a disease ordisorder characterized by the overexpression of a small-noncoding RNA ina subject, comprising administering to the subject anHES-oligonucleotide complex, containing an oligonucleotide which istargeted to nucleic acids comprising or encoding the small non-codingRNA and which acts to reduce the levels of the small non-coding RNAand/or interfere with its function in the subject. In some embodiments,the HES-oligonucleotide is an anti-miRNA (anti-miR) In additionalembodiments the anti-miRNA is double stranded. In further embodiments,the HES-oligonucleotide contains an anti-miRNA that is double strandedand contains oligonucleotides of 18-23 units in length and is bluntended or comprises one or more 3′ overhangs of 1, 2, or 3 nucleotides.In additional embodiments, the HES-oligonucleotide contains a singlestranded anti-miR that is 8-25 units in length. HES-oligonucleotidescontaining expression vectors that express these anti-MiRs are alsoencompassed by the invention. In some embodiments, the oligonucleotidecomprises a sequence substantially complementary to the overexpressedsmall-noncoding RNA.

In further embodiments, the invention encompasses a method of treating adisease or disorder characterized by the overexpression of a miRNA in asubject comprising administering to the subject an HES-oligonucleotidecomplex containing an oligonucleotide which is targeted to nucleic acidscomprising or encoding the miRNA and which acts to reduce the levels ofthe miRNA and/or interfere with its function in the subject. In someembodiments, the oligonucleotide comprises a sequence substantiallycomplementary to the overexpressed miRNA.

a. Families of miRNAs can be characterized by nucleotide identity atpositions 2-8 of the miRNA, a region known as the seed sequence. Themembers of a miRNA family are herein termed “related miRNAs”. Eachmember of a miRNA family shares an identical seed sequence that plays anessential role in miRNA targeting and function. As used herein, the term“seed sequence” or “seed region” refers to nucleotides 2 to 9 from the5′-end of a mature miRNA sequence. Examples of miRNA families are knownin the art and include, but are not limited to, the let-7 family (having9 miRNAs), the miR-15 family (comprising miR-15a, miR-15b, miR15-16,miR-16-1, and miR-195), and the miR-181 family (comprising miR-181a,miR-181b, and miR-181c). In some embodiments, an HES-oligonucleotidespecifically hybridizes to the seed region of a miRNA and interfereswith the processing or function of the miRNA. In some embodiments, theHES-oligonucleotide specifically hybridizes to the seed region of amiRNA and interferes with the processing or function of multiple miRNAs.In further embodiments, at least 2 of the multiple miRNAs have relatedseed sequences or are members of the miRNA superfamily.

The association of miRNA dysfunction with diseases such as cancer,fibrosis, metabolic disorders and inflammatory disorders and the abilityof miRNAs to influence an entire network of genes involved in a commoncellular process makes the selective modulation of miRNAs usinganti-miRNAs and miRNA mimics particularly attractive disease modulatingtherapeutics. The invention also encompasses a method of treating adisease or disorder characterized by the overexpression of a protein ina subject, comprising administering to the subject anHES-oligonucleotide complex, containing an oligonucleotide which istargeted to nucleic acids comprising or encoding a small non-coding RNAthat influences the increased production of the protein, wherein theoligonucleotide act to reduce the levels of the small non-coding RNAand/or interfere with its function in the subject. In some embodiments,the oligonucleotide comprises a sequence substantially complementary tothe small-noncoding RNA.

The invention also encompasses a method of treating a disease ordisorder characterized by the overexpression of a protein in a subject,comprising administering to the subject an HES-oligonucleotide complex,containing an oligonucleotide which is targeted to nucleic acidscomprising or encoding a miRNA that influences the increased productionof the protein, wherein the oligonucleotide acts to reduce the levels ofthe miRNA and/or interfere with its function in the subject. In someembodiments, the oligonucleotide comprises a sequence substantiallycomplementary (specifically hybridizable) to the miRNA.

The invention also encompasses a method of treating a disease ordisorder characterized by the under expression of a small-noncoding RNAin a subject, comprising administering to the subject anHES-oligonucleotide complex, containing an oligonucleotide whichcomprises or encodes the small non-coding RNA or increases theendogenous expression, processing or function of the small non-codingRNA, and which acts to increase the level of the small non-coding RNAand/or increase its function in the subject. In some embodiments, theoligonucleotide comprises a sequence substantially complementaryspecifically hybridizable) to the overexpressed small-noncoding RNA.

In further embodiments, the invention encompasses a method of treating adisease or disorder characterized by the overexpression of a miRNA in asubject, comprising administering to the subject an HES-oligonucleotidecomplex, containing an oligonucleotide which comprises or encodes thesmall non-coding RNA or increases the endogenous expression, processingor function of the small non-coding RNA, and which acts to increase thelevel of the small non-coding RNA and/or increase its function in thesubject. In some embodiments, the oligonucleotide comprises a sequencesubstantially complementary to the overexpressed miRNA.

The invention also encompasses a method of treating a disease ordisorder characterized by the overexpression of a protein in a subject,comprising administering to the subject an HES-oligonucleotide complex,containing an oligonucleotide which comprises or encodes the smallnon-coding RNA or increases the endogenous expression, processing orfunction of the small non-coding RNA, and which acts to increase thelevel of the small non-coding RNA and/or increase its function in thesubject. In some embodiments, the oligonucleotide comprises a sequencesubstantially complementary to the small-noncoding RNA.

The invention also encompasses a method of treating a disease ordisorder characterized by the overexpression of a protein in a subject,comprising administering to the subject an HES-oligonucleotide complex,containing an oligonucleotide which comprises or encodes the smallnon-coding RNA or increases the endogenous expression, processing orfunction of the small non-coding RNA, and which acts to increase thelevel of the small non-coding RNA and/or increase its function in thesubject. In some embodiments, the oligonucleotide comprises a sequencesubstantially complementary (specifically hybridizable) to the miRNA.

In another embodiment, the invention provides a method of inhibitingmiRNA activity comprising administering to subject anHES-oligonucleotide complex having anti-miRNA activity, such as thosedescribed herein.

In some embodiments, the HES-oligonucleotide complex contains anoligonucleotide selected from: a siRNA, a miRNA, a dicer substrate(e.g., dsRNA), a ribozyme, a decoy, an aptamer, an antisenseoligonucleotide and a plasmid capable of expressing an siRNA, a miRNA,or an antisense oligonucleotide.

In some embodiments, the oligonucleotides are chimeric oligonucleotidescomprising an internal region containing at least 1, at least 2, atleast 3, at least 4, at least 5, or all 2′-F modified nucleotides andexternal regions comprising at least one stability enhancingmodifications. In one embodiment, an oligonucleotide in theHES-oligonucleotide complex comprises an internal region having a first2′-modified nucleotide and external regions each comprising a second2′-modified nucleotide. In a further embodiment, the gap regioncomprises one or more 2′-fluoro modifications and the wing regionscomprise one or more 2′-methoxyethyl modifications. In one embodiment,the oligonucleotide in the HES-oligonucleotide complex is ISIS 393206 orISIS 327985.

Therapeutic

Diagnostics, Drug Discovery and Therapeutics

The oligonucleotides, complexes and other compositions of the inventionhave uses that include, but are not limited to, research, drugdiscovery, kits and diagnostics, and therapeutics. The complexes of theinvention are particularly suited for use in in vivo methods due totheir improved oligonucleotide delivery over conventional deliverytechniques.

The invention provides compositions and methods for detecting a nucleicacid sequence in vitro or in vivo. Thus, in some embodiments, theinvention provides compositions comprising an HES-oligonucleotidecomplex containing an oligonucleotide that specifically hybridizes witha target nucleic acid under physiologic conditions.

In some embodiments, an HES-oligonucleotide delivery vehicle of theinvention is used to identify the presence of an infectious agent in ahost organism such as a virus in a mammalian cell or a bacterium in amammalian tissue. In this embodiment an HES-oligonucleotide which iscomposed of an HES, serves as an in vivo marker of binding to acomplementary sequence. This identification is accomplished by thedetection of changes in fluorescence when binding of theHES-oligonucleotide to a complementary foreign (e.g., infectious agent)nucleic acid sequence results in destruction or significant loss of theHES and results in a loss of fluorescence quenching. Thus, the inventionencompasses methods for determining the presence of, and/or quantitatingthe levels of, a foreign nucleic acid in a host organism (subject). Insome embodiments, the method is performed in vitro. In otherembodiments, the method is performed in vivo.

In some embodiments, the invention provides a method for detecting thepresence of an infectious agent in a subject in vitro or in vivo,comprising, contacting a cell, tissue or subject with anHES-oligonucleotide containing an oligonucleotide that specificallyhybridizes with the nucleic acid of an infectious agent, determining thelevel of fluorescence in the cell, tissue or subject tissue, andcomparing said level of fluorescence with that obtained for a controlcell, tissue or subject not containing the infectious agent that hasbeen contacted with the HES-oligonucleotide, wherein an increasedfluorescence compared to the control indicates that the cell, tissue, orsubject has the infectious agent.

In additional embodiments, an HES-oligonucleotide of the invention isused to identify an altered level of a nucleic acid that is a biomarkerfor a disease or disorder. In some embodiments, the invention provides amethod for detecting the presence of an altered level of a nucleic acidbiomarker for a disease or disorder in vitro comprising, contacting acell or tissue with an HES-oligonucleotide containing an oligonucleotidethat specifically hybridizes with the nucleic acid biomarker,determining the level of fluorescence in the cell or tissue andcomparing said level of fluorescence with that obtained for a controlcell or tissue that has been contacted with the HES-oligonucleotide,wherein an altered fluorescence compared to the control indicates thatthe cell or tissue has an altered level of the nucleic acid biomarker.

In further embodiments, the invention provides a method for detecting analtered level of a nucleic acid biomarker for a disease or disorder invivo comprising, administerng to a subject an HES-oligonucleotidecontaining an oligonucleotide that specifically hybridizes with thenucleic acid biomarker, determining the level of fluorescence in thesubject, and comparing said level of fluorescence with that obtained fora control subject that has been administered the HES-oligonucleotide,wherein an altered fluorescence compared to the control indicates thatthe subject has an altered level of the nucleic acid biomarker. Thisapproach can also be used to quantitate the number of copies of anaberrant gene of host origin in vivo.

In vitro and in vivo fluorescence can be monitored using techniquesknown to those skilled in the art. For example, in some embodiments,fluorescence is monitored via fluorescence endoscopy. Fluorescenceendoscopy can be performed using equipment such as, the Olympus EVISExERA-II CLV-80 system (Olympus Corp., Tokyo Japan) using theappropriate excitation wavelengths and the emission filters for theadministered fluorphores. Fluorescence intensities can be determinedusing techniques and software known in the art such as, the Image-Jsoftware (NIH, Bethesda, Md.).

In some embodiments, the disease or disorder is: cancer, fibrosis, aproliferative disease or disorder, a neurological disease or disorder,and inflammatory disease or disorder, a disease or disorder of theimmune system, a disease or disorder of the cardiovascular system, ametabolic disease or disorder, a disease or disorder of the skeletalsystem, or a disease or disorder of the skin or eyes. In additionalembodiments, the disease or disorder is a disease or disorder of thekidneys, liver, lymph nodes, spleen or adipose tissue. In particularembodiments, the disease or disorder is not a disease or disorder of thekidneys, liver, lymph nodes, spleen or adipose tissue.

In further embodiments, the disease or disorder is a proliferativedisorder such as, cancer. For example, the overexpression of numerousmiRNA such, as mIR-10b, mIR17-92, mIR-21, mIR125b, mIR-155, mIR193a,mIR-205a and mIR-210, have been associated with various forms of cancer.In some embodiments, the biomarker is a miRNA selected from mIR-10b,mIR17-92, mIR-21, mIR125b, mIR-155, mIR193a, mIR-205a, and mIR-210, andan increased fluorescence of the cell, tissue, or subject relative to acontrol indicates that the subject has cancer or has a predispositionfor cancer.

In additional embodiments, the methods of the invention are used toidentify and/or distinguish between different diseases or disorders. Themethods of the invention can likewise be used to determine among otherthings, altered nucleic acid (e.g., DNA and RNA) profiles thatdistinguish between normal and diseased (e.g., cancerous) tissue orcells, discriminate between different subtypes of diseased cells (e.g.,between different cancers and subtypes of a particular cancer), todiscriminate between mutations (e.g., oncogenic mutations) giving riseto or associated with different disease states, and to identify tissuesof origin (e.g., in a metastasized tumor).

Moreover, in some embodiments, the oligonucleotides in theHES-oligonucleotides of the invention are therapeutic oligonucleotides,and the destruction or significant loss of HES that results in anincreased fluorescence when the therapeutic HES oligonucleotidesspecifically hybridizes with target nucleic acids indicates that thetherapeutic oligonucleotides have been delivered to, and have hybridizedwith the target nucleic acid. Thus, in some embodiments, the inventionprovides a method for monitoring and/or quantitating the delivery of atherapeutic oligonucleotide to a target nucleic acid in vivo, comprisingadministering to a subject, a HES oligonucleotides containing atherapeutic oligonucleotide that specifically hybridizes to the targetnucleic acid, and determining the level of fluorescence in a cell ortissue of the subject, wherein an increased fluorescence in the cell ortissue compared to a control cell or tissue indicates that that thetherapeutic oligonucleotide has been delivered to and hybridized withthe target nucleic acid.

The delivery vehicles of the invention are based, in part, on thesurprising discovery that the linking of one or more HES to a single ormultiple strands of oligonucleotides significantly enhances the in vivodelivery of the HES-oligonucleotides inside a cell or tissue of a liveorganism. Thus, the HES-oligonucleotide vehicles of the invention haveapplications as therapeutic delivery vehicles for a broad range oftherapeutic applications as well as in conjunction with assays andtherapies to evaluate for example, the activity and/or number of copiesof a specific gene or RNA in vivo.

For use in research and drug discovery, an HES-oligonucleotide of theinvention can be used for example, to interfere with the normal functionof the nucleic acid molecules to which they are targeted. Expressionpatterns within cells, tissues, or subjects treated with one or moreHES-oligonucleotides of the invention are then compared to controlcells, tissues or subjects not treated with the compounds and thepatterns produced are then analyzed for differential levels of nucleicacid and/or protein expression and as they pertain, for example, todisease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds that affect expressionpatterns.

The invention also provides compositions and methods for modulatingnucleic acids and protein encoded or regulated by these modulatednucleic acids. In particular embodiments, the invention providescompositions and methods for modulating the levels, expression,processing or function of a mRNA, small non-coding RNA (e.g., miRNA), agene or a protein.

In some embodiments, the invention provides a method of delivering anoligonucleotide to a cell in vivo by administering to a subject anHES-oligonucleotide complex containing the oligonucleotide. Inparticular embodiments, the oligonucleotide is a therapeuticoligonucleotide.

Thus, in some embodiments, the invention encompasses compositions, suchas pharmaceutical compositions, comprising an HES-oligonucleotidecomplex having at least one oligonucleotide hybridizable with a targetnucleic, acid sequence under physiologic conditions.

In some embodiments, the invention provides a method of delivering anoligonucleotide to a subject In particular embodiments, the inventionprovides a method of delivering a therapeutic oligonucleotide to asubject comprising administering an HES-oligonucleotide complex to thesubject, wherein the complex contains a therapeutically effective amountof an oligonucleotide sufficient to modulate a target RNA (e.g., mRNAand miRNA) or target gene.

According to one embodiment, the invention provides a method ofmodulating a target nucleic acid in a subject comprising administeringan HES-oligonucleotide complex to the subject., wherein anoligonucleotide of the complex comprises a sequence substantiallycomplementary to the target nucleic acid that specifically hybridizes toand modulates levels of the nucleic acid or interferes with itsprocessing or function. In some embodiments, the target nucleic acid isRNA, in further embodiments the RNA is mRNA or miRNA. In furtherembodiments, the oligonucleotide reduces the level of a target RNA by atleast 10%, at least 20%, at least 30%, at least 40% or at least 50% inone or more cells or tissues of the subject. In some embodiments, thetarget nucleic acid is a DNA.

According to one embodiment, the invention provides a method ofmodulating a protein in a subject comprising, administering anHES-oligonucleotide complex to the subject, wherein an oligonucleotideof the complex comprises a sequence substantially complementary to anucleic acid that encodes the protein or influences the transcription,translation, production, processing or function of the protein. In someembodiments, the oligonucleotide specifically hybridizes to an RNA. Infurther embodiments the RNA is mRNA or miRNA. In additional embodiments,the oligonucleotide reduces the level of the protein or RNA by at least10%, at least 20%, at least 30%, at least 40% or at least 50% in one ormore cells or tissues of the subject. In some embodiments, theoligonucleotide specifically hybridizes to a DNA.

In particular embodiments, the oligonucleotide in theHES-oligonucleotide complex is selected from an siRNA, an shRNA, amiRNA, an anti-miRNA, a dicer substrate (e.g., dsRNA), an aptamer, adecoy, an antisense oligonucleotide, and a plasmid capable of expressingan siRNA, a miRNA, or an antisense oligonucleotide. In some embodiments,the oligonucleotide specifically hybridizes with an RNA or a sequenceencoding an RNA. In other embodiments, the oligonucleotide specifcallyhybridizes with DNA sequence encoding an RNA or the regulatory sequencesthereof.

In additional embodiments, the expression of a nucleic acid or proteinis modulated in a subject by contacting the subject with anHES-oligonucleotide complex containing an antisense oligonucleotide. Inparticular embodiments, the antisense oligonucleotide in theHES-oligonucleotide complex is a substrate for RNAse H when bound to atarget RNA. In some embodiments, the antisense oligonucleotide is agapmer. As used herein, a “gapmer” refers an antisense compound having acentral region (also referred to as a “gap” or “gap segment”) positionedbetween two external flanking regions (also referred to as “wings” or“wing segments”). The regions are differentiated by the types of sugarmoieties comprising each distinct region. The types of sugar moietiesthat are used to differentiate the regions of a gapmer may in someembodiments include beta-D-ribonucleosides, beta-D-deoxyribonucleosides,2′-modified nucleosides (such 2′-modified nucleosides may include2′-MOE, 2′-fluoro and 2′-O—CH3, among others), and bicyclic sugarmodified nucleosides (such bicyclic sugar modified nucleosides mayinclude LNA™ or ENA™, among others).

In some embodiments, each wing of a gapmer oligonucleotides comprisesthe same number of subunits. In other embodiments, one wing of a gapmeroligonucleotide comprises a different number of subunits than the otherwing of the gapmer. In one embodiment, the wings of gapmeroligonucleotides have, independently, from 1 to about 5 nucleosides ofwhich, 1, 2 3 4 or 5 of the wing nucleosides are sugar modifiednucleosides. In one embodiment, the central or gap region contains 8-25beta-D-ribonucleosides or beta-D-deoxyribonucleosides (i.e., is 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24 or 25 nucleosidesin length). In a further embodiment, the central or gap region contains17-24 nucleotides (i.e., is 17, 18, 19, 20, 21, 22, 23 or 24 nucleosidesin length). In some embodiments, the gapmer oligonucleotide comprisesphosphodiester internucleotide linkages, phosphorothioateinternucleotide linkages, or a combination of phosphodiester andphosphorothioate internucleotide linkages. In particular embodiments thecentral region of the gapmer oligonucleotide contains at least 2, 3, 4,5 or 10 modified nucleosides, modified internucleoside linkages orcombinations thereof. In particular embodiments the central region ofthe gapmer oligonucleotide contains at least 10beta-D-2′-deoxy-2′-fluororibofuranosyl nucleosides. In some embodiments,each nucleoside in the central region of the oligonucleotide abeta-D-2′-deoxy-2′-fluororibofuranosyl nucleoside. In one embodiment,the gapmer oligonucleotides is fully complementary over the lengthcomplementarity with the target RNA. In one embodiment, one or bothwings of the gapmer contains at least one 2′ modified nucleoside. In oneembodiment, one or both wings of the gapmer contains 1, 2 or 3 2′-MOEmodified nucleosides. In one embodiment, one or both wings of the gapmercontains 1, 2 or 3 2′-OCH3 modified nucleosides. In another embodiment,one or both wings of the gapmer contains 1, 2 or 3 LNA or alpha-LNAnucleosides. In some embodiments, the LNA or alpha LNA in the wings ofthe gapmer contain one or more methyl groups in the (R) or (S)configuration at the 6′ (2′,4′-constrained-2′-O-ethyl BNA, S-cEt) or the5′-position (-5′-Me-LNA or -5′-Me-alpha LNA) of LNA or alternativelycontain a substituted carbon atom in place of the 2′-oxygen atom in theLNA or alpha LNA. In further embodiments, the LNA or alpha LNA in thegapmer contain a steric bulk moiety at the 5′ position (e.g., a methylgroup). In a further embodiment, the gap comprises at least one 2′fluoro modified nucleosides. In an additional embodiment, the wings areeach 2 or 3 nucleosides in length and the gap region is 19 nucleotidesin length. In additional embodiments, the gapmer has at least one5-methylcytosine.

In another embodiment, the nucleosides of the central region (gap)contain uniform sugar moieties that are different than the sugarmoieties in one or both of the external wing regions. In onenon-limiting example, the gap is uniformly comprised of a first2′-modified nucleoside and each of the wings is uniformly comprised of asecond 2′-modified nucleoside. For example, in one embodiment, thecentral region contains 2′-F modified nucleotides flanked on each end byexternal regions each having two 2′-MOE modified nucleotides(2′-MOE/2′-F/2′-MOE). In particular embodiments, the gapmer is ISIS393206. In another embodiment, the central region contains 2′-F modifiednucleotides flanked on each end by external regions each having two2′-MOE modified nucleotides (2′-MOE/2′-F/2 WOE). In particularembodiments, the external regions each having two LNA or alpha LNAmodified nucleotides in the wings of the gapmer. In further embodiments,the LNA or alpha LNA modified nucleotides contain one or more methylgroups in the (R) or (S) configuration at the 6′(2′,4′-constrained-2′-O-ethyl BNA, S-cEt) or the 5′-position (-5′-Me-LNAor -5′-Me-alpha LNA) of LNA or alternatively contain a substitutedcarbon atom in place of the 2′-oxygen atom in the LNA or alpha LNA.

In another embodiment, the invention provides for the use of anHES-oligonucleotide complex of the invention in the manufacture of acomposition for the treatment of one or more of the conditionsassociated with a miRNA or an miRNA family.

According to one embodiment, the methods comprise the step ofadministering to or contacting the subject with an effective amount ofan HES-oligonucleotide of the invention sufficient to modulate thetarget gene or RNA (e.g., mRNA and miRNA) expression and to therebytreat one or more conditions or symptoms associated with the disease ordisorder. Exemplary compounds of the invention effectively modulate theexpression, activity or function of the gene, mRNA or small-non-codingRNA target. In preferred embodiments, the small non-coding RNA target isa miRNA, a pre-miRNA, or a polycistronic or monocistronic pri-miRNA. Inadditional embodiments, the small non-coding RNA target is a singlemember of a miRNA family. In a further embodiment, two or more membersof a miRNA family are selected for modulation.

In an additional embodiment, the invention provides a method ofinhibiting the activity of a target nucleic acid in a subject,comprising administering to the subject an HES-oligonucleotide complexcomprising an oligonucleotide which is targeted to nucleic acidscomprising or encoding the nucleic acid and which acts to reduce thelevels of the nucleic acid and/or interfere with its function in thecell. In particular embodiments, the target nucleic acid is a small-noncoding RNA, such as, a miRNA. In some embodiments, the oligonucleotidecomprises a sequence substantially complementary to the target nucleicacid.

In some embodiments, some embodiments, the invention provides a methodof reducing expression of a target RNA in an subject in need of reducingexpression of said target RNA, comprising administering to said subjectan antisense HES-oligonucleotide complex. In particular embodiments, anoligonucleotide in the complex is a substrate for RNAse H when bound tosaid target mRNA. In some embodiments, the oligonucleotide is a gapmer.

In an additional embodiment of the present invention is a method ofreducing expression of a target RNA in a subject in need of reducingexpression of said target RNA, comprising administering to said subjecta HES-oligonucleotide complex containing an antisense oligonucleotide tosaid subject wherein the antisense sequence specifically hybridizes tothe target RNA. In particular embodiments, the antisense oligonucleotidein the HES-oligonucleotide complex is a substrate for RNAse H when boundto a target RNA. In additional embodiments, the antisenseoligonucleotide is a gapmer. In some embodiments, the oligonucleotide is18 to 24 nucleotides in length comprising: a gap region having greaterthan 11 contiguous 2′-deoxyribonucleotides; and a first wing region anda second wing region flanking the gap region, wherein each of said firstand second wing regions independently have 1 to 82′-O-(2-methoxyethyl)ribonucleotides.

In another embodiment, the antisense oligonucleotide is not a substratefor RNAse H when bound to the target RNA (e.g., mRNA and miRNA). In someembodiments, the oligonucleotide comprises at least one modified sugarmoiety comprising a modification at the 2′-position. In someembodiments, each nucleoside of the oligonucleotide comprises a modifiedsugar moiety comprising a modification at the 2′-position. In someembodiments the oligonucleotide comprises at least one PNA motif. Infurther embodiments, all the monomeric units of the oligonucleotidecorrespond to a PNA. In other embodiments the oligonucleotide comprisesat least one morpholino motif. In some embodiments, the morpholino is aphosphorodiamidate morpholino. In further embodiments, all the monomericunits of the oligonucleotide correspond to a morpholino. In furtherembodiments, all the monomeric units of the oligonucleotide correspondto a phosphorodiamidate morpholino (e.g., PMO). In some embodiments, theoligonucleotide sequence is specifically hybridizable to a sequencewithin 30 nucleotides of the AUG start codon of the target RNA. Inadditional embodiments, the HES-oligonucleotide sequence is specificallyhybridizable to a sequence in the 5′ untranslated region of the targetRNA. In some embodiments, the HES-oligonucleotides are designed totarget the 3′ untranslated sequence in an RNA (e.g., mRNA). In furtherembodiments, the HES-oligonucleotides are designed to target the 3′untranslated sequence in an RNA that is bound by an miRNA (i.e., themiRNA 3′UTR target site in an mRNA). One such example is “miR-Mask” or“target protector,” which are single-stranded 2′-O-methyl-modified (orother chemically modified) antisense oligonucleotide fully complementaryto predicted miRNA binding sites in the 3′-UTR of a specific targetmRNA, covering up the access of the miRNA to its binding site on thetarget mRNA (see, e.g., Choi et al (2007) Science 318:271; Wang (2011)Methods Mol. Biol. 676:43). In further embodiments, theHES-oligonucleotides are designed to mimic the 3′ untranslated sequencein an mRNA that is bound by an miRNA. One such example is “miRNAsponges,” competitive miRNA inhibitory transgene expressing multipletandem binding sites for an endogenous miRNA, which stably interact withthe corresponding miRNA and prevent the association of target miRNA withits endogenous target mRNAs. In additional embodiments, the nucleic acidis an mRNA and the oligonucleotide sequence is specifically hybridizableto a target region of a RNA selected from the group consisting of: anintron/exon junction of a target RNA, an intron/exon junction and aregion 1 to 50 nucleobases 5′ of an intron/exon junction of the targetRNA. In some embodiments, the target region is selected from the groupconsisting of: a region 1 to 15 nucleobases 5′ of an intron/exonjunction, 20 to 24 nucleobases 5′ of an intron/exon junction, and 30 to50 nucleobases 5′ of an intron/exon junction. In further embodiments,the HES-oligonucleotide complex contains an oligonucleotide thatspecifically hybridizes to nucleotides 1-10 of a miRNA (i.e., the seedregion) or that specifically hybridizes to a sequence in aprecursor-miRNA (pre-miRNA) or primary-miRNA (pri-miRNA) that when boundby the oligonucleotide blocks miRNA processing.

In another embodiment, the invention provides a method of inhibiting theproduction of a protein, comprising administering to a subject anHES-oligonucleotide complex containing an oligonucleotide which istargeted to nucleic acids encoding the protein or decreases theendogenous expression, processing or function of the protein in thesubject. In some embodiments, the oligonucleotide comprises a sequencesubstantially complementary to a nucleic acid encoding the protein.

In some embodiments, the invention provides a method of decreasing theamount of a target cellular RNA or corresponding protein in a cell bycontacting a cell expressing the target RNA with an HES-oligonucleotidecomplex having an oligonucleotide sequence that specifically hybridizesto the target RNA, wherein the amount of the target RNA or correspondingprotein is reduced. In some embodiments, the RNA is an mRNA or a miRNA.In additional embodiments the oligonucleotide is selected from a siRNA,a shRNA, a miRNA, a anti-miRNA, a dicer substrate (e.g., dsRNA), adecoy, an aptamer, a decoy, an antisense oligonucleotide and a plasmidcapable of expressing an siRNA, a miRNA, a anti-miRNA, a ribozyme or anantisense oligonucleotide.

In particular embodiments, the oligonucleotide in theHES-oligonucleotide is an antisense oligonucleotide. In one embodiment,the antisense oligonucleotide is a substrate for RNAse H when bound to atarget RNA. In additional embodiments, the antisense oligonucleotide isa gapmer. In some embodiments, the oligonucleotide is 18 to 24nucleotides in length comprising: a gap region having greater than 11contiguous 2′-deoxyribonucleotides; and a first wing region and a secondwing region flanking the gap region, wherein each of said first andsecond wing regions independently have 1 to 82′-O-(2-methoxyethyl)ribonucleotides. In particular embodiments, theoligonucleotide contains 12 to 30 linked nucleosides.

In another embodiment, the oligonucleotide is not a substrate for RNAseH when bound to the target RNA (e.g., mRNA and miRNA). In someembodiments, the oligonucleotide comprises at least one modified sugarmoiety comprising a modification at the 2′-position. In someembodiments, each nucleoside of the oligonucleotide comprises a modifiedsugar moiety comprising a modification at the 2′-position. In someembodiments the oligonucleotide comprises at least one PNA motif. Infurther embodiments, all the monomeric units of the oligonucleotidecorrespond to a PNA. In other embodiments the oligonucleotide comprisesat least one morpholino motif. In a further embodiment theoligonucleotide comprises at least one phosphorodiamidate morpholino. Infurther embodiments, all the monomeric units of the oligonucleotidecorrespond to a morpholino. In further embodiments, all the monomericunits of the oligonucleotide correspond to a phosphorodiamidatemorpholino (PMO). In some embodiments, the oligonucleotide sequencespecifically hybridizes to a sequence within 30 nucleotides of the AUGstart codon of the target RNA. In some embodiments, theHES-oligonucleotides are designed to target the 3′ untranslated sequencein an RNA (e.g., mRNA). In further embodiments, the HES-oligonucleotidesare designed to target the 3′ untranslated sequence in an RNA that isbound by an miRNA. In additional embodiments, the target RNA is mRNA andthe oligonucleotide sequence specifically hybridizes to a target regionof the mRNA selected from the group consisting of an intron/exonjunction of a target RNA, and an intron/exon junction and a region 1 to50 nucleobases 5′ of an intron/exon junction of the target RNA. In someembodiments, the target region is selected from the group consisting of:a region 1 to 15 nucleobases 5′ of an intron/exon junction, 20 to 24nucleobases 5′ of an intron/exon junction, and 30 to 50 nucleobases 5′of an intron/exon junction. In further embodiments, theHES-oligonucleotide complex contains an oligonucleotide thatspecifically hybridizes to nucleotides 1-10 of a miRNA (i.e., the seedregion) or that specifically hybridizes to a sequence in aprecursor-miRNA (pre-miRNA) or primary-miRNA (pri-miRNA) that when boundby the oligonucleotide blocks miRNA processing.

In some embodiments, the oligonucleotide can induce RNA interference(RNAi). In some embodiments the oligonucleotide is siRNA, shRNA or aDicer substrate. In some embodiments, the oligonucleotide is a siRNAthat is 18-35 nucleotides in length. In some embodiments, theoligonucleotide is an shRNA that has a stem of 19 to 29 nucleotides inlength and a loop size of between 4-30 nucleotides. In furtherembodiments the siRNA or shRNA oligonucleotide contains one or moremodified nucleosides, modified internucleoside linkages, or combinationsthereof. In some embodiments, the oligonucleotide is a Dicer substrateand contains 2 nucleic acid strands that are each 18-25 nucleotides inlength and contain a 2 nucleotide 3′ overhang. In particularembodiments, the Dicer substrate is a double stranded nucleic acidcontaining 21 nucleotides in length and contains a two nucleotide 3′overhang. In further embodiments one or both strands of the Dicersubstrate contains one or more modified nucleosides, modifiedinternucleoside linkages, or combinations thereof.

In additional embodiments, the invention provides a method of reducingthe expression of a target RNA in a subject in need of such reducedexpression of the target RNA, comprising administering to the subject anHES-oligonucleotide complex having an oligonucleotide sequence thatspecifically hybridizes to the target RNA, wherein the expression of thetarget RNA in a cell or tissue of the subject is reduced. In someembodiments, the RNA is an mRNA or a miRNA. In additional embodimentsthe oligonucleotide is selected from a siRNA, shRNA, miRNA, ananti-miRNA, a dicer substrate, an aptamer, a decoy, an antisenseoligonucleotide, a plasmid capable of expressing a siRNA, a miRNA, aribozyme and an antisense oligonucleotide.

In particular embodiments, the oligonucleotide in theHES-oligonucleotide is an antisense oligonucleotide. In one embodiment,the antisense oligonucleotide is a substrate for RNAse H when bound tothe target RNA (e.g., mRNA and miRNA). In additional embodiments, theantisense oligonucleotide is a gapmer. In some embodiments, theoligonucleotide is 18 to 24 nucleotides in length comprising: a gapregion having greater than 11 contiguous 2′-deoxyribonucleotides; and afirst wing region and a second wing region flanking the gap region,wherein each of said first and second wing regions independently have 1to 8 2′-O-(2-methoxyethyl)ribonucleotides. In particular embodiments,the oligonucleotide contains 12 to 30 linked nucleosides.

In another embodiment, the antisense oligonucleotide is not a substratefor RNAse H when bound to the target RNA (e.g., mRNA and miRNA). In someembodiments, the oligonucleotide comprises at least one modified sugarmoiety comprising a modification at the 2′-position. In someembodiments, each of the nucleosides of the oligonucleotide comprise amodified sugar moiety comprising a modification at the 2′-position. Insome embodiments the oligonucleotide comprises at least one PNA motif.In further embodiments, all the monomeric units of the oligonucleotidecorrespond to a PNA. In other embodiments the oligonucleotide containsat least one morpholino motif. En some embodiments, the morpholino is aphosphorodiamidate morpholino. In further embodiments, all the monomericunits of the oligonucleotide correspond to a morpholino. In furtherembodiments, all the monomeric units of the oligonucleotide correspondto a phosphorodiamidate morpholino (PMO). In some embodiments, theoligonucleotide sequence specifically hybridizes to a sequence within 30nucleotides of the AUG start codon of the target RNA. In additionalembodiments, the oligonucleotide sequence specifically hybridizes to asequence in the 5′ untranslated region of the target RNA. In someembodiments, the HES-oligonucleotides are designed to target the 3′untranslated sequence in an RNA (e.g., mRNA). In further embodiments,the HES-oligonucleotides are designed to target the 3′ untranslatedsequence in an RNA that is bound by an miRNA. In additional embodiments,the target RNA is mRNA and the oligonucleotide sequence specificallyhybridizes to a target region of the target mRNA selected from the groupconsisting of: an intron/exon junction of a target RNA, and anintron/exon junction and a region 1 to 50 nucleobases 5′ of anintron/exon junction of the target RNA. In some embodiments, the targetregion is selected from the group consisting of: a region 1 to 15nucleobases 5′ of an intron/exon junction, 20 to 24 nucleobases 5′ of anintron/exon junction, and 30 to 50 nucleobases 5′ of an intron/exonjunction. In further embodiments, the HES-oligonucleotide complexcontains an oligonucleotide that specifically hybridizes to nucleotides1-10 of a miRNA (i.e., the seed region) or that specifically hybridizesto a sequence in a precursor-miRNA (pre-miRNA) or primary-miRNA(pri-miRNA) that when bound by the oligonucleotide blocks miRNAprocessing.

In some embodiments, the oligonucleotide can induce RNA interference(RNAi). In some embodiments the oligonucleotide is siRNA, shRNA or aDicer substrate. In some embodiments, the oligonucleotide is a siRNAthat is 18-35 nucleotides in length. In some embodiments, theoligonucleotide is an shRNA that has a stem of 19 to 29 nucleotides inlength and a loop size of between 4-30 nucleotides. In furtherembodiments the siRNA or shRNA oligonucleotide contains one or moremodified nucleosides, modified internucleoside linkages, or combinationsthereof. In some embodiments, the oligonucleotide is a Dicer substrateand contains 2 nucleic acid strands that are each 18-25 nucleotides inlength and contain a 2 nucleotide 3′ overhang. In particularembodiments, the Dicer substrate is a double stranded nucleic acidcontaining 21 nucleotides in length and contains a two nucleotide 3′overhang. In further embodiments one or both strands of the Dicersubstrate contains one or more modified nucleosides, modifiedinternucleoside linkages, or combinations thereof.

In some embodiments, an HES-oligonucleotide complex is administered to asubject to deliver an oligonucleotide that specifically hybridizes to atarget nucleic acid (e.g., gene, mRNA or miRNA), which provides a growthadvantage for a tumor cell or enhances the replication of amicroorganism. In other embodiments, an HES-oligonucleotide complex isadministered to deliver an antisense, siRNA, shRNA, Dicer substrate ormiRNA targeting an mRNA sequence coding for a protein (e.g., a proteinvariant) which has been implicated in a disease. Thus, in someembodiments, the invention provides an in vivo delivery system fortransporting specific nucleic acid sequences into live cells to forexample, silence genes in organisms afflicted with pathologic conditionsdue to aberrant gene expression.

In some embodiments, the invention provides a method of decreasing theamount of a polypeptide of interest in a cell, comprising: contacting acell expressing a nucleic acid that encodes the polypeptide, or acomplement thereof, with an HES-oligonucleotide complex having anoligonucleotide sequence specifically hybridizes to a DNA or mRNAencoding the polypeptide, such that the expression of the polypeptide ofinterest is reduced. In further embodiments the oligonucleotide isselected from a siRNA, shRNA, miRNA, an anti-miRNA, a dicer substrate,an antisense oligonucleotide, a plasmid capable of expressing a siRNA, amiRNA, a ribozyme and an antisense oligonucleotide, and wherein theoligonucleotide specifically hybridizes to a nucleic acid that encodesthe polypeptide, or a complement thereof, such that the expression ofthe polypeptide is reduced. In particular embodiments, theoligonucleotide contains 12 to 30 linked nucleosides. In someembodiments, the complex contains a double-stranded RNA (dsRNA). In someembodiments, the oligonucleotide comprises at least one modifiedoligonucleotide. In further embodiments, the oligonucleotide comprisesat least one modified oligonucleotide motif selected from a 2′modification (e.g., 2′-fluoro, 2′-OME and 2′-methoxyethyl (2′-MOE)) alocked nucleic acid (LNA and alpha LNA), a PNA motif, and morpholinomotif.

In particular embodiments, the oligonucleotide in theHES-oligonucleotide complex is antisense sequence and is a substrate forRNAse H when bound to a target RNA. In additional embodiments, theantisense oligonucleotide is a gapmer. In some embodiments, the gapmeris an antisense oligonucleotide that is a chimeric oligonucleotide. Insome embodiments, the chimeric oligonucleotide comprises a2′-deoxynucleotide central gap region positioned between 5′ and 3′ wingsegments. The wing segments contain nucleosides containing at least one2′-modified sugar. The wing segments are contain nucleosides containingat least one 2′ sugar moiety selected from a 2′-O-methoxyethyl sugarmoiety or a bicyclic nucleic acid sugar moiety. In some embodiments, thegap segment may be ten 2′-deoxynucleotides in length and each of thewing segments may be five 2′-O-methoxyethyl nucleotides in length. Thechimeric oligonucleotide may be uniformly comprised of phosphorothioateinternucleoside linkages. Further, each cytosine of the chimericoligonucleotide may be a 5′-methylcytosine.

In another embodiment, the antisense oligonucleotide is not a substratefor RNAse H when hybridized to the RNA. In some embodiments, eachnucleoside of the oligonucleotide comprises a modified sugar moietycomprising a modification at the 2′-position. In some embodiments theoligonucleotide contains at least one PNA motif. In further embodiments,all the monomeric units of the oligonucleotide correspond to a PNA. Inother embodiments the oligonucleotide contains at least one morpholinomotif. In some embodiments, the morpholino is a phosphorodiamidatemorpholino. In further embodiments, all the monomeric units of theoligonucleotide correspond to a morpholino. In further embodiments, allthe monomeric units of the oligonucleotide correspond to aphosphorodiamidate morpholino (PMO). In some embodiments, theoligonucleotide sequence specifically hybridizes to a sequence within 30nucleotides of the AUG start codon of the target RNA. In additionalembodiments, the oligonucleotide sequence specifically hybridizes to asequence in the 5′ untranslated region of the target RNA. In someembodiments, the HES-oligonucleotides are designed to target the 3′untranslated sequence in an RNA (e.g., mRNA). In further embodiments,the HES-oligonucleotides are designed to target the 3′ untranslatedsequence in an RNA that is bound by an miRNA. In additional embodiments,the oligonucleotide sequence specifically hybridizes to a target regionof a target mRNA selected from the group consisting of an intron/exonjunction of a target RNA, and an intron/exon junction and a region 1 to50 nucleobases 5′ of an intron/exon junction of the target RNA. In someembodiments, the target region is selected from the group consisting of:a region 1 to 15 nucleobases 5′ of an intron/exon junction, 20 to 24nucleobases 5′ of an intron/exon junction, and 30 to 50 nucleobases 5′of an intron/exon junction. In further embodiments, theHES-oligonucleotide complex contains an oligonucleotide thatspecifically hybridizes to nucleotides 1-10 of a miRNA (i.e., the seedregion) or that specifically hybridizes to a sequence in aprecursor-miRNA (pre-miRNA) or primary-miRNA (pri-miRNA) that when boundby the oligonucleotide blocks miRNA processing.

In further embodiments, the oligonucleotide can induce RNA interference(RNAi). In some embodiments the oligonucleotide is siRNA, shRNA or aDicer substrate. in some embodiments, the oligonucleotide is an siRNAthat is 18-35 nucleotides in length. In some embodiments, theoligonucleotide is an shRNA that has a stem of 19 to 29 nucleotides inlength and a loop size of between 4-30 nucleotides. In furtherembodiments the siRNA or shRNA oligonucleotide contains one or moremodified nucleosides, modified internucleoside linkages, or combinationsthereof. In some embodiments, the oligonucleotide is a Dicer substrateand contains 2 nucleic acid strands that are each 18-25 nucleotides inlength and contain a 2 nucleotide 3′ overhang. In particularembodiments, the Dicer substrate is a double stranded nucleic acidcontaining 21 nucleotides in length and contains a two nucleotide 3′overhang. In further embodiments one or both strands of the Dicersubstrate contains one or more modified nucleosides, modifiedinternucleoside linkages, or combinations thereof.

In an additional embodiment, the invention provides a method ofincreasing the activity of a nucleic acid in a subject, comprisingadministering to the subject an HES-oligonucleotide complex containingan oligonucleotide which comprises or encodes the nucleic acid orincreases the endogenous expression, processing or function of thenucleic acid (e.g., by binding regulatory sequences in the gene encodingthe nucleic acid) and which acts to increase the level of the nucleicacid and/or increase its function in the cell. In some embodiments, theoligonucleotide comprises a sequence substantially the same as nucleicacids comprising or encoding the nucleic acid.

In another embodiment, the invention provides a method of increasing theproduction of a protein, comprising administering to a subject anHES-oligonucleotide complex containing an oligonucleotide which encodesthe protein or increases the endogenous expression, processing orfunction of the protein in the subject. In some embodiments, theoligonucleotide comprises a sequence substantially the same as nucleicacids encoding the protein. In some embodiments, the oligonucleotideshares 100% identity with at least 15 contiguous nucleotides, at least20 contiguous nucleotides or over the fall-length of an endogenousnucleic acid sequence encoding the protein.

The invention also encompasses a method of treating a disease ordisorder characterized by the overexpression of a nucleic acid in asubject, comprising administering to the subject an HES-oligonucleotidecomplex containing an oligonucleotide which is targeted to a nucleicacid comprising or encoding the nucleic acid and which acts to reducethe levels of the nucleic acid and/or interfere with its function in thesubject. In some embodiments, the nucleic acid is DNA, mRNA or miRNA. Inadditional embodiments the oligonucleotide is selected from an siRNA, anshRNA, a miRNA, an anti-miRNA, a dicer substrate, an antisenseoligonucleotide, a plasmid capable of expressing an siRNA, a miRNA, aribozyme and an antisense oligonucleotide.

In particular embodiments, the nucleic acid is RNA and theoligonucleotide in the HES-oligonucleotide is an antisenseoligonucleotide. In one embodiment, the antisense oligonucleotide is asubstrate for RNAse H when hybridized to the RNA. In additionalembodiments, the antisense oligonucleotide is a gapmer. In someembodiments, the oligonucleotide is 18 to 24 nucleotides in lengthcomprising: a gap region having greater than 11 contiguous2′-deoxyribonucleotides; and a first wing region and a second wingregion flanking the gap region, wherein each of said first and secondwing regions independently have 1 to 82′-O-(2-methoxyethyl)ribonucleotides. In particular embodiments, theoligonucleotide contains 12 to 30 linked nucleosides. In someembodiments, the oligonucleotide comprises a sequence substantiallycomplementary to the nucleic acid.

In another embodiment, the oligonucleotide is not a substrate for RNAseH when bound to the nucleic acid. In some embodiments, each nucleosideof the oligonucleotide comprises a modified sugar moiety comprising amodification at the 2′-position. In some embodiments the oligonucleotidecontains at least one PNA motif. In further embodiments, all themonomeric units of the oligonucleotide correspond to a PNA. In otherembodiments the oligonucleotide contains at least one morpholino motif.In some embodiments, the morpholino is a phosphorodiamidate morpholino.In further embodiments, all the monomeric units of the oligonucleotidecorrespond to a morpholino. In further embodiments, all the monomericunits of the oligonucleotide correspond to a phosphorodiamidatemorpholino (PMO). In some embodiments, the oligonucleotide sequencespecifically hybridizes to a sequence within 30 nucleotides of the AUGstart codon of the target RNA. In additional embodiments, theoligonucleotide sequence specifically hybridizes to a sequence in the 5′untranslated region of the target RNA. In some embodiments, theHES-oligonucleotides are designed to target the 3′ untranslated sequencein an RNA (e.g., mRNA). In further embodiments, the HES-oligonucleotidesare designed to target the 3′ untranslated sequence in an RNA that isbound by an miRNA. In additional embodiments, the nucleic acid is mRNAand the oligonucleotide sequence specifically hybridizes to a targetregion of the mRNA selected from the group consisting of: an intron/exonjunction of a target RNA, and an intron/exon junction and a region 1 to50 nucleobases 5′ of an intron/exon junction of the target RNA. In someembodiments, the target region is selected from the group consisting of:a region 1 to 15 nucleobases 5′ of an intron/exon junction, 20 to 24nucleobases 5′ of an intron/exon junction, and 30 to 50 nucleobases 5′of an intron/exon junction. In further embodiments, theHES-oligonucleotide complex contains an oligonucleotide thatspecifically hybridizes to nucleotides 1-10 of a miRNA (i.e., the seedregion) or that specifically hybridizes to a sequence in aprecursor-miRNA (pre-miRNA) or primary-miRNA (pri-miRNA) that when boundby the oligonucleotide blocks miRNA processing.

In further embodiments, the oligonucleotide can induce RNA interference(RNAi). In some embodiments the oligonucleotide is siRNA, shRNA or aDicer substrate. In some embodiments, the oligonucleotide is an siRNAthat is 18-35 nucleotides in length. In some embodiments, theoligonucleotide is an shRNA that has a stem of 19 to 29 nucleotides inlength and a loop size of between 4-30 nucleotides. In furtherembodiments the siRNA or shRNA oligonucleotide contains one or moremodified nucleosides, modified internucleoside linkages, or combinationsthereof. In some embodiments, the oligonucleotide is a Dicer substrateand contains 2 nucleic acid strands that are each 18-25 nucleotides inlength and contain a 2 nucleotide 3¹ overhang. In particularembodiments, the Dicer substrate is a double stranded nucleic acidcontaining 21 nucleotides in length and contains a two nucleotide 3′overhang. In further embodiments one or both strands of the Dicersubstrate contains one or more modified nucleosides, modifiedinternucleoside linkages, or combinations thereof.

In further embodiments, the invention encompasses a method of treating adisease or disorder characterized by the overexpression of a protein ina subject, comprising administering to the subject anHES-oligonucleotide complex containing an oligonucleotide which istargeted to a nucleic acid encoding the protein or decreases theendogenous expression, processing or function of the protein in thesubject. In some embodiments, the nucleic acid is DNA, mRNA or miRNA. Inadditional embodiments the oligonucleotide is selected from an siRNA, anshRNA, miRNA, an anti-miRNA, a dicer substrate, an aptamer, a decoy, anantisense oligonucleotide, a plasmid capaLle of expressing an siRNA, anmiRNA, a ribozyme and an antisense oligonucleotide. In some embodiments,the oligonucleotide shares 100% identity with at least 15 contiguousnucleotides, at least 20 contiguous nucleotides or over the full-lengthof an endogenous nucleic acid sequence encoding the protein.

In particular embodiments, the targeted nucleic acid is RNA and theoligonucleotide in the HES-oligonucleotide is an antisenseoligonucleotide. In one embodiment, the antisense oligonucleotide is asubstrate for RNAse H when hybridized to the RNA. In additionalembodiments, the antisense oligonucleotide is a gapmer. In someembodiments, the oligonucleotide is 18 to 24 nucleotides in lengthcomprising: a gap region having greater than 11 contiguous2′-deoxyribonucleotides; and a first wing region and a second wingregion flanking the gap region, wherein each of said first and secondwing regions independently have 1 to 82′-O-(2-methoxyethyl)ribonucleotides. In particular embodiments, theoligonucleotide contains 12 to 30 linked nucleosides. In someembodiments, the oligonucleotide comprises a sequence substantiallycomplementary to the nucleic acid.

In another embodiment, the oligonucleotide is not a substrate for RNAseH when bound to the target RNA (e.g., mRNA and miRNA). In someembodiments, the oligonucleotide comprises at least one modified sugarmoiety comprising a modification at the 2′-position. In someembodiments, each nucleoside of the oligonucleotide comprises a modifiedsugar moiety comprising a modification at the 2′-position. In someembodiments the oligonucleotide comprises at least one PNA motif. Infurther embodiments, all the monomeric units of the oligonucleotidecorrespond to a PNA. In other embodiments the oligonucleotide comprisesat least one morpholino motif. In some embodiments, the morpholino is aphosphorodiamidate morpholino. Iii further embodiments, all themonomeric units of the oligonucleotide correspond to a morpholino. Infurther embodiments, all the monomeric units of the oligonucleotidecorrespond to a phosphorodiamidate morpholino (PMO). In someembodiments, the oligonucleotide sequence specifically hybridizes to asequence within 30 nucleotides of the AUG start codon of the target RNA.In additional embodiments, the oligonucleotide sequence is specificallyhybridizable to a sequence in the 5′ untranslated region of the targetRNA. (e.g., within 30 nucleotides of the AUG start codon) and to reducetranslation. In some embodiments, the HES-oligonucleotides are designedto target the 3′ untranslated sequence in an RNA (e.g., mRNA). Infurther embodiments, the HES-oligonucleotides are designed to target the3′ untranslated sequence in an RNA that is bound by an miRNA. Inadditional embodiments, the nucleic acid is mRNA and the oligonucleotidesequence specifically hybridizes to a target region of an mRNA encodingthe protein selected from the group consisting of: an intron/exonjunction of a target RNA, and an intron/exon junction and a region 1 to50 nucleobases 5′ of an intron/exon junction of the target RNA. In someembodiments, the target region is selected from the group consisting of:a region 1 to 15 nucleobases 5′ of an intron/exon junction, 20 to 24nucleobases 5′ of an intron/exon junction, and 30 to 50 nucleobases 5′of an intron/exon junction. In further embodiments, theHES-oligonucleotide complex contains an oligonucleotide thatspecifically hybridizes to nucleotides 1-10 of a miRNA (i.e., the seedregion) or that specifically hybridizes to a sequence in aprecursor-miRNA (pre-miRNA) or primary-miRNA (pri-miRNA) that when boundby the oligonucleotide blocks miRNA processing.

In further embodiments, the oligonucleotide can induce RNA interference(RNAi). In some embodiments the oligonucleotide is siRNA, shRNA or aDicer substrate. In some embodiments, the oligonucleotide is an siRNAthat is 18-35 nucleotides in length. In some embodiments, theoligonucleotide is an shRNA that has a stem of 19 to 29 nucleotides inlength and a loop size of between 4-30 nucleotides. In furtherembodiments the siRNA or shRNA oligonucleotide contains one or moremodified nucleosides, modified internucleoside linkages, or combinationsthereof. In some embodiments, the oligonucleotide is a Dicer substrateand contains 2 nucleic acid strands that are each 18-25 nucleotides inlength and contain a 2 nucleotide 3′ overhang. In particularembodiments, the Dicer substrate is a double stranded nucleic acidcontaining 21 nucleotides in length and contains a two nucleotide 3′overhang. In further embodiments one or both strands of the Dicersubstrate contains one or more modified nucleosides, modifiedinternucleoside linkages, or combinations thereof.

The invention also encompasses a method of treating (e.g., alleviating)a disease or disorder characterized by the aberrant expression of aprotein in a subject, comprising administering to the subject anHES-oligonucleotide complex, containing an oligonucleotide whichspecifically hybridizes to the mRNA encoding the protein and alter thesplicing of the target RNA (e.g., promoting exon skipping). In someembodiments, each nucleoside of the oligonucleotide comprises at leastone modified sugar moiety comprising a modification at the 2′-position.In particular embodiments, the modified oligonucleotide is a 2′ OME or2′ allyl. In additional embodiments, the modified oligonucleotide isLNA, alpha LNA (e.g., an LNA or alpha LNA containing a steric bulkmoiety at the 5′ position (e.g., a methyl group). In some embodimentsthe oligonucleotide contains at least one PNA motif. In furtherembodiments, all the monomeric units of the oligonucleotide correspondto a PNA. In other embodiments the oligonucleotide contains at least onemorpholino motif. In some embodiments, the morpholino is aphosphorodiamidate morpholino. In further embodiments, all the monomericunits of the oligonucleotide correspond to a morpholino. In furtherembodiments, all the monomeric units of the oligonucleotide correspondto a phosphorodiamidate morpholino (PMO). In some embodiments, theoligonucleotide sequence specifically hybridizes to a sequence within 30nucleotides of the AUG start codon of the target RNA. In additionalembodiments, the oligonucleotide sequence specifically hybridizes to asequence in the 5′ untranslated region of the target RNA. In someembodiments, the HES-oligonucleotides are designed to target the 3′untranslated sequence in an RNA (e.g., mRNA). In further embodiments,the HES-oligonucleotides are designed to target the 3′ untranslatedsequence in an RNA that is bound by an miRNA. In additional embodiments,oligonucleotide sequence is specifically hybridizable to a target regionof an mRNA selected from the group consisting of: an intron/exonjunction of a target RNA, and an intron/exon junction and a region 1 to50 nucleobases 5′ of an intron/exon junction of the target RNA. In someembodiments, the target region is selected from the group consisting of:a region 1 to 15 nucleobases 5′ of an intron/exon junction, 20 to 24nucleobases 5′ of an intron/exon junction, and 30 to 50 nucleobases 5′of an intron/exon junction.

In particular embodiments, the disease or disorder is Duchenne MuscularDystrophy (DMD). In some embodiments, the oligonucleotide specificallyhybridizes to mRNA sequence that promotes message splicing to “skipover” exon 44, 45, 50, 51, 52, 53 or 55 of the dystrophin gene. Inparticular embodiments, the oligonucleotide specifically hybridizes tomRNA sequence that promotes message splicing to “skip over” exon 51 ofthe dystrophin gene. In particular embodiments, the oligonucleotide inthe HES-oligonucleotide complex is AVI-4658 (AVI Biopharma). In otherembodiments, the oligonucleotide in the HES-oligonucleotide complex iscompetes for dystrophin mRNA binding with AVI-4658.

A farther embodiment of the invention provides a method comprising,selecting a subject who has received a diagnosis of a disease ordisorder, administering to the subject a therapeutically effectiveamount of a HES-oligonucleotide complex containing an oligonucleotidethat specifically hybridizes to a nucleic acid sequence believed to beassociated with or to encode a protein associated with the disease ordisorder or a condition related thereto, and monitoring diseaseprogression in the subject.

In some embodiments, the nucleic acid is DNA, mRNA or miRNA. Inadditional embodiments the oligonucleotide is selected from an siRNA, anshRNA, a miRNA, an anti-miRNA, a dicer substrate, an aptamer, a decoy,an antisense oligonucleotide, a plasmid capable of expressing an siRNA,a miRNA, a ribozyme and an antisense oligonucleotide. In someembodiments, the oligonucleotide shares 100% identity with at least 15contiguous nucleotides, at least 20 contiguous nucleotides or over thefull-length of the nucleic acid.

In particular embodiments, the nucleic acid is RNA and theoligonucleotide in the HES-oligonucleotide is an antisenseoligonucleotide. In one embodiment, the antisense oligonucleotide is asubstrate for RNAse H when hybridized to the RNA. In additionalembodiments, the antisense oligonucleotide is a gapmer. In someembodiments, the oligonucleotide is 18 to 24 nucleotides in lengthcomprising: a gap region having greater than 11 contiguous2′-deoxyribonucleotides; and a frst wing region and a second wing regionflanking the gap region, wherein each of said first and second wingregions independently have 1 to 8 2′-O-(2-methoxyethyl)ribonucleotides.In particular embodiments, the oligonucleotide contains 12 to 30 linkednucleosides. In some embodiments, the oligonucleotide comprises asequence substantially complementary to the nucleic acid.

In another embodiment, the oligonucleotide is not a substrate for RNAseH when bound to the target RNA (e.g., mRNA and miRNA). In someembodiments, the oligonucleotide comprises at least one modified sugarmoiety comprising a modification at the 2′-position. In someembodiments, all the nucleosides of the oligonucleotide comprise amodified sugar moiety comprising a modification at the 2′-position. Insome embodiments the oligonucleotide comprises at least one PNA motif.In further embodiments, all the monomeric units of the oligonucleotidecorrespond to a PNA. In other embodiments the oligonucleotide comprisesat least one morpholino motif. In some embodiments, the morpholino is aphosphorodiamidate morpholino. In additional embodiments, all themonomeric units of the oligonucleotide correspond to a morpholino. Infarther embodiments all the monomeric units of the oligonucleotidecorrespond to a phosphorodiamidate morpholino (PMO). In someembodiments, the oligonucleotide sequence specifically hybridizes to asequence within 30 nucleotides of the AUG start codon of the target RNA.In additional embodiments, the oligonucleotide sequence specificallyhybridizes to a sequence in the 5′ untranslated region of the targetRNA. In some embodiments, the HES-oligonucleotides are designed totarget the 3′ untranslated sequence in an RNA (e.g., mRNA). In furtherembodiments, the HES-oligonucleotides are designed to target the 3′untranslated sequence in an RNA that is bound by an miRNA. In additionalembodiments, the oligonucleotide specifically hybridizes to a targetregion of the mRNA selected from the group consisting of: an intron exonjunction of a target RNA, and an intron/exon junction and a region 1 to50 nucleobases 5′ of an intron/exon junction of the target RNA. In someembodiments, the target region is selected from the group consisting ofa region 1 to 15 nucleobases 5′ of an intron/exon junction, 20 to 24nucleobases 5′ of an intron/exon junction, and 30 to 50 nucleobases 5′of an intron/exon junction. In additional embodiments, theHES-oligonucleotide complex contains an oligonucleotide thatspecifically hybridizes to nucleotides 1-10 of a miRNA (i.e., the seedregion) or that specifically hybridizes to a sequence in aprecursor-miRNA (pre-miRNA) or primary-miRNA (pri-miRNA) that when boundby the oligonucleotide blocks miRNA processing.

In further embodiments, the oligonucleotide can induce RNA interference(RNAi). In some embodiments the oligonucleotide is siRNA, shRNA or aDicer substrate. In some embodiments, the oligonucleotide is an siRNAthat is 18-35 nucleotides in length. In some embodiments, theoligonucleotide is an shRNA that has a stem of 19 to 29 nucleotides inlength and a loop size of between 4-30 nucleotides. In furtherembodiments the siRNA or shRNA oligonucleotide contains one or moremodified nucleosides, modified internucleoside linkages, or combinationsthereof in some embodiments, the oligonucleotide is a Dicer substrateand contains 2 nucleic acid strands that are each 18-25 nucleotides inlength and contain a 2 nucleotide 3′ overhang. In particularembodiments, the Dicer substrate is a double stranded nucleic acidcontaining 21 nucleotides in length and contains a two nucleotide 3′overhang. In further embodiments one or both strands of the Dicersubstrate contains one or more modified nucleosides, modifiedinternucleoside linkages, or combinations thereof.

In another embodiment, the invention provides a method of slowingdisease progression in a subject suffering from a disease or disordercorrelated with the overexpression of a protein comprising,administering to the subject an HES-oligonucleotide complex containingan oligonucleotide that specifically hybridizes to a DNA or mRNAencoding the protein, such that the expression of the polypeptide isreduced. In additional embodiments the oligonucleotide is selected froman siRNA, an shRNA, a miRNA, an anti-miRNA, a dicer substrate, anantisense oligonucleotide, a plasmid capable of expressing an siRNA, amiRNA, a ribozyme and an antisense oligonucleotide. In some embodiments,the oligonucleotide shares 100% identity with at least 15 contiguousnucleotides, at least 20 contiguous nucleotides or over the full-lengthof the DNA or mRNA encoding the protein.

In particular embodiments, the nucleic acid is mRNA and theoligonucleotide in the HES-oligonucleotide is an antisenseoligonucleotide. In one embodiment, the antisense oligonucleotide is asubstrate for RNAse H when hybridized to the RNA. In additionalembodiments, the antisense oligonucleotide is a gapmer. In someembodiments, the oligonucleotide is 18 to 24 nucleotides in lengthcomprising: a gap region having greater than 11 contiguous2′-deoxyribonucleotides; and a first wing region and a second wingregion flanking the gap region, wherein each of said first and secondwing regions independently have 1 to 82′-O-(2-methoxyethyl)ribonucleotides. In particular embodiments, theoligonucleotide contains 12 to 30 linked nucleosides. In someembodiments, the oligonucleotide comprises a sequence substantiallycomplementary to the nucleic acid.

In another embodiment, the oligonucleotide is not a substrate for RNAseH when bound to the target RNA (e.g., mRNA and miRNA). In someembodiments, the oligonucleotide comprises at least one modified sugarmoiety comprising a modification at the 2′-position. In someembodiments, each nucleoside of the oligonucleotide comprises a modifiedsugar moiety comprising a modification at the 2′-position. In someembodiments the oligonucleotide comprises at least one PNA motif. Infurther embodiments, all the monomeric units of the oligonucleotidecorrespond to a PNA. In other embodiments the oligonucleotide comprisesat least one morpholino motif. In some embodiments, the morpholino is aphosphorodiamidate morpholino. In further embodiments, all the monomericunits of the oligonucleotide correspond to a morpholino. In furtherembodiments, all the monomeric units of the oligonucleotide correspondto a phosphorodiamidate morpholino (PMO). In some embodiments, theoligonucleotide sequence specifically hybridizes to a sequence within 30nucleotides of the AUG start codon of the target RNA. In additionalembodiments, the oligonucleotide sequence is specifically hybridizableto a sequence in the 5′ untranslated region of the target RNA. In someembodiments, the HES-oligonucleotides are designed to target the 3′untranslated sequence in an RNA (e.g., mRNA). In further embodiments,the HES-oligonucleotides are designed to target the 3′ untranslatedsequence in an RNA that is bound by an miRNA. In additional embodiments,the nucleic acid is an mRNA and the oligonucleotide sequencespecifically hybridizes to a target region of the mRNA selected from thegroup consisting of: an intron/exon junction of a target RNA, and anintron/exon junction and a region 1 to 50 nucleobases 5′ of anintron/exon junction of the target RNA. In some embodiments, the targetregion is selected from the group consisting of: a region 1 to 15nucleobases 5′ of an intron/exon junction, 20 to 24 nucleobases 5′ of anintron/exon junction, and 30 to 50 nucleobases 5′ of an intron/exonjunction.

In further embodiments, the oligonucleotide can induce RNA interference(RNAi). In some embodiments the oligonucleotide is siRNA, shRNA or aDicer substrate. In some embodiments, the oligonucleotide is an siRNAthat is 18-35 nucleotides in length. In some embodiments, theoligonucleotide is an shRNA that has a stem of 19 to 29 nucleotides inlength and a loop size of between 4-30 nucleotides. In furtherembodiments the siRNA or shRNA oligonucleotide contains one or moremodified nucleosides, modified internucleoside linkages, or combinationsthereof. In some embodiments, the oligonucleotide is a Dicer substrateand contains 2 nucleic acid strands that are each 18-25 nucleotides inlength and contain a 2 nucleotide 3′ overhang. In particularembodiments, the Dicer substrate is a double stranded nucleic acidcontaining 21 nucleotides in length and contains a two nucleotide 3′overhang. In further embodiments one or both strands of the Dicersubstrate contains one or more modified nucleosides, modifiedinternucleoside linkages, or combinations thereof.

Therapeutic Applications on miRNA-Related Pathologies

There currently exist several distinct groups of pathological conditionsthat are known to be regulated by an miRNA or a family of miRNA, whichcan be targeted using the HES-oligonucleotide complexes of the presentinvention.

In one embodiment, an oligonucleotide in an HES-oligonucleotide complexis an inhibitor or mimic of one or more miRNAs associated with aninfectious disease. In one embodiment, an oligonucleotide in theHES-oligonucleotide complex of the invention inhibits miR-122.Miravirsen (SPC3649), an inhibitor of miR-122 developed by SantarisPharma A/S. Mir-122 is a liver specific miRNA that the Hepatitis C virusrequires for replication as a critical endogenous host factor. Clinicaltrial data for 4-week Miravirsen monotherapy has shown robustdose-dependent anti-viral activity. Regulus Therapeutics andGlaxoSmithKline (GSK) have likewise demonstrated in a preclinical studythat miR-122 is essential in the replication of HCV and plan to advancean anti-miR-122 into clinical studies for the treatment of HCVinfection.

In another embodiment, an oligonucleotide in an HES-oligonucleotidecomplex is an inhibitor or mimic of an miRNA associated with fibrosis.In one embodiment, an oligonucleotide in the HES-oligonucleotide complexof the invention inhibits miR-21. Preclinical studies by RegulusPharmaceutical and Sanofi Aventis have shown that inhibition of miR-21,which is upregulated in human fibrotic tissues, can improve organfunction in multiple models of fibrosis including heart and kidney. Inanother embodiment, an oligonucleotide in the HES-oligonucleotidecomplex of the invention corresponds to or mimics miR-29. MGN-4220,mimics or miRNA replacement therapy by Mirna Therapeutics, targetsmiR-29 implicated in cardiac fibrosis.

In another embodiment, an oligonucleotide in an HES-oligonucleotidecomplex is an inhibitor or mimic of an miRNA associated with acardiovascular disease, including, but not limited to, stroke, heartdisease, atherosclerosis, restenosis, thrombosis, anemia, leucopenia,neutropenia, thrombocytopenia, granuloctopenia, pancytoia and idiopathicthrombocytopenic purpura. In one embodiment, an oligonucleotide in theHES-oligonucleotide complex of the invention inhibits miR-33. RegulusPharmaceutical and AstraZeneca has shown in preclinical studies that theinhibition of miR-33 reduces arterial plaque size and increase levels ofHDL. In another embodiment, an oligonucleotide in theHES-oligonucleotide complex of the invention inhibits miR-92, miR-378,miR-206 and/or the miR-143/145 family. MGN-6114, MGN-5804, MGN-2677,MGN-8107, developed by Miragen Therapeutics, respectively targets miR-92implicated in peripheral arterial disease, miR-378 implicated incardiometablolic disease, miR-143/145 family implicated in vasculardisease, and miR-206 implicated in amylotrophic lateral sclerosis. In afurther embodiment, an oligonucleotide in the HES-oligonucleotidecomplex of the invention inhibits the miR-208/209 family and/or themiR-15/195 family. Miragen Therapeutics's MGN-9103 and MGN-1374 aremiRNA inhibitors that respectively target miR-208/209 family for chronicheart failure and miR-15/195 family for post-myocardial infarctionremodeling. In another embodiment, an oligonucleotide in theHES-oligonucleotide complex of the invention inhibits miR-126 and/ormiR92a. miR-126 and miR-92a play central roles in the development of anatherosclerotic plaque.

In another embodiment, an oligonucleotide in the HES-oligonucleotidecomplex is an inhibitor of an miRNA associated with a neurologicaldisease or conditions. In one embodiment, an oligonucleotide in theHES-oligonucleotide complex of the invention inhibits miR-206, miR-206plays a crucial role in ALS and in neuromuscular synapse regeneration.

In another embodiment, an oligonucleotide in the HES-oligonucleotidecomplex is an inhibitor or mimic of an miRNAs associated withoncological conditions. In one embodiment, an oligonucleotide in theHES-oligonucleotide complex of the invention inhibits miR-21. miR-21 hasbeen suggested by numerous scientific publications to play an importantrole in the initiation and progression of cancers including liver,kidney, breast, prostate, lung and brain. Anti-miR-21 in hepatocellularcarcinoma (HCC) mouse model has shown delayed tumor progression in apreclinical study by Regulus Pharmaceutical and Sanofi Aventis. Inanother embodiment, an oligonucleotide in the HES-oligonucleotidecomplex of the invention inhibits miR-10b. Preclinical animal studies ofanti-miR-10b by Regulus Pharmaceutical also showed therapeutic effect inGBM model. In an additional embodiment, an oligonucleotide in theHES-oligonucleotide complex of the invention corresponds to or mimicsmiR-34. Mimics or miRNA replacement therapy by Mirna Therapeutics ofmiR-34, which is lost or expressed at reduced levels in most solid andhematologic malignancies, showed inhibition of growth for various typesof cancers in preclinical studies of MRX34.

In some embodiments, an oligonucleotide in the HES-oligonucleotidecomplex is an inhibitor of an miRNAs selected from: let-7a, miR-9,miR-10b, miR-15a-miR-16-1, miR-16, miR-21, miR-24, miR-26a, miR-34a,miR-103-107, miR-122, miR-133, miR-181, miR-192, miR-194, miR-200. ThesemicroRNAs are among those that have been reported to be associated withcancer.

In some embodiments, an oligonucleotide in the HES-oligonucleotidecomplex inhibits a miRNA selected from: let-7, let-7a, let-7f, miR-1,Mir-10b, miR-15a-miR-16-1, Mir-17-5p, Mir-17-92, miR-21, Mir-23-27,miR-25, miR-27b, miR-29, miR-30a, Mir-31, miR-34a, miR-92-1, miR-106a,miR-125, Mir-126, Mir-130a, Mir-132, miR-133b, Mir-155, miR-206,Mir-210, Mir-221/222, miR-223, Mir-296, miR-335, Mir-373, Mir-378,miR-380-5p, Mir-424, miR-451, miR-486-5p, and Mir-520c. These microRNAsare among those that have been reported to promote neovascularization,metastasis and/or the onset of cancer.

In some embodiments, an oligonucleotide in the HES-oligonucleotidecomplex inhibits a miRNA selected from: miR-15 family, miR-21, miR-23,miR-24, miR-27, miR-29, miR-33, miR-92a, miR-145, miR-155, miR-199b,miR-208a/b family, miR-320, miR-328, miR-499. These microRNAs are amongthose that have been reported to have various roles in cardiovascularfunctions.

In some embodiments, an oligonucleotide in the HES-oligonucleotidecomplex inhibits a miRNA selected from: let-7b, miR-9, miR106b-25cluster, miR-124, miR-132, miR-137, miR-184. These microRNAs are amongthose that have been reported to have various roles in adultneurogenesis in neural stem cells (NSCs).

In some embodiments, an oligonucleotide in the HES-oligonucleotidecomplex is an inhibitor or mimic of an miRNAs selected from: let-7a,miR-21, mir-26, miR-125b, mir-145, miR-155, miR-191, miR-193a, miR-200family, miR-205, miR-221, and miR-222. These microRNAs are among thosethat have been reported to function as diagnostic or prognosticbiomarkers for various types of cancers. In particular embodiment, anoligonucleotide in the HES-oligonucleotide complex is an inhibitor of amiRNA selected from: miR-21, mir-26, miR-125b, miR-155, miR-193a,miR-200 family, miR-221, and miR-222. In particular embodiment, anoligonucleotide in the HES-oligonucleotide complex contains the sequenceof, or mimics a miRNA selected from: let-7a, mir-145, miR-191, andmiR-205.

In some embodiments, an oligonucleotide in the HES-oligonucleotidecomplex is an inhibitor of an miRNAs selected from: miR-138, mir-182,miR-21, mir-103/107, miR-29c. These microRNAs are among those that havebeen reported to have roles in arthritis, lupus, atherosclerosis,insulin sensitivity, and albuminuria, respectively.

In some embodiments, an oligonucleotide in the HES-oligonucleotidecomplex is an inhibitor or mimic of an miRNAs selected from: let-7,let-7-a3, lin-28, miR-1, miR-9-1, miR-15a, miR-16-1, miR-17-92 cluster,miR-21, miR-29 family, miR-34 family, miR-124, miR-127, and miR-290.These microRNAs are among those that have been reported to bedysregulated in various types of cancers due to abnormalities in geneticor epigenetic regulations responsible for miRNA expression. Inparticular embodiment, an oligonucleotide in the HES-oligonucleotidecomplex is an inhibitor of a miRNA selected from: let-7-a3, lin-28,miR-17-92 cluster, and miR-21. In particular embodiment, anoligonucleotide in the HES-oligonucleotide complex contains the sequenceof, or mimics a miRNA selected from: let-7, miR-1, miR-15a, miR-16-1,miR-21, miR-29 family, miR-34 family, miR-124, miR-127, and miR-290.

In further embodiments, an oligonucleotide in the HES-oligonucleotidecomplex contains the sequence of, or mimics an miRNA selected from:Mir-20a, Mir-34, Mir-92a, Mir-200c, Mir-217 and Mir-503. These miRNAsare among those that have been reported to be antiangiogenic.

In an additional embodiment, an oligonucleotide in theHES-oligonucleotide complex of the invention contains the sequence of ormimics: miR-1, miR-2, miR-6, miR-7 or let-7. In particular embodiments,the oligonucleotides are miR-Rx07, miR-Rx06, miR-Rxlet-7, miR-Rx01,miR-Rx02 or miR-Rx03. In an additional embodiment, an oligonucleotide inthe HES-oligonucleotide complex of the invention corresponds to ormimics miR-451, miR-451 has been demonstrated to regulate erythropoiesisin vivo (Patrick et al.; Genes & Dev., 2010) and thus to be implicatedin diseases such as, polycythemia vera, red cell dyscrasias generally,or other hematopoietic malignancies. In particular embodiments, theoligonucleotide is MGN-4893.

In additional embodiments, pharmaceutical compositions comprising anantisense compound targeted to a nucleic acid of interest are used forthe preparation of a composition for treating a patient suffering orsusceptible to a disease or disorder associated with the nucleic acid.

Ex Vivo Delivery of miRNAs for Nuclear Reprogramming and Generation ofiPSCs

In additional embodiments, the invention provides a method for cellnuclear reprograming. In some embodiments, an HES-oligonucleotidescontaining one or more mimics and/or inhibitor of a miRNA or a pluralityof miRNAs are administered ex vivo into cells such as, human and mousesomatic cells to reprogram the cells to have one or more properties ofinduced pluripotent stem cells (iPSCs) or embryonic stem (ES)-likepluripotent cells (e.g., colony morphology of induced iPSC and embryoidbody (EB), expression of stem cell marker genes in the reprogrammed stemcell lines shown by qRT-PCR, hematoxylin and eosin staining of teratomasderived from iPSC clones showing pluripotency of forming mesoderm,endoderm, and ectoderm, immunohistochemistry analysis of iPSC-derivedteratoma tissues showing expression of germ layer-specificdifferentiation markers, teratoma formation upon transplantation intoSCID mouse). The non-toxic and highly efficient HES-oligonucleotidedelivery system of the invention provides a greatly increased efficiencyof delivery method for reprogramming cells compared to conventionaloligonucleotide delivery methods (see, e.g., U.S. Publ. Nos.2010/0075421, US 2009/0246875, US 2009/0203141, and US 2008/0293143).

Examples of miRNAs or mimics of miRNAs that can be administered tosomatic cells according to the methods of the present invention andthereby induce reprogramming of the somatic cells to display one or moreproperties of iPSC include a miRNA or miRNA mimic of a miRNA selectedfrom: lin-28, miR-17-92 cluster, miR-93, miR-106b, miR-106b-25 cluster,miR-106a-363 cluster, miR-181a, miR-199b, miR-200c, miR-214, miR-302,miR-367, miR-302-367 cluster, miR-369, miR-371, miR-372, miR-373, andmiR-520, as well as the family members and variants of these miRNAs(see, e.g., Anokye-Danso et al. (2011) Cell Stem Cell 8, 376; Miyoshi etal. (2011) Cell Stem Cell 8, 1; Subramanyam et al. (2011) NatureBiotechnology, 29:5; Li et al. (2011) The EMBO Journal 30:5; Lin et al.(2011) Nucleic Acids Research 39:3; Lakshmipathy et al (2010)Regenerative Medicine 5:4; Xu et al. (2009) Cell 137:647; Goff et al.(2009) PLoS One 4:9; Wilson et al. (2009) Stem Cells Dev. 18:5; Chin etal. (2009) Cell Stem Cell 5:1; Ren et al. (2009) Journal ofTranslational Medicine 7:20; Lin et al. (2008) RNA 14:2115, the contentsof each of which is hereby incorporated by reference in its entirety).Examples of inhibitors of miRNAs that can be administered to somaticcells according to the methods of the present invention and therebyinduce reprogramming of the somatic cells to display one or moreproperties of iPSC include an inhibitor of a miRNA selected from: let-7,miR-145, as well as the family members and variants of these miRNAs(see, e.g., Lakshmipathy et al (2010) Regenerative Medicine 5:4; Xu etal. (2009) Cell 137:647, the contents of each of which is herebyincorporated by reference in its entirety). In further embodiments, theinvention encompasses a method of inducing the reprogramming of somaticcells comprising administering to the cells HES-oligonucleotidescontaining a miRNA, miRNA mimic or miRNA inhibitor of 1, 2, 3, 4, 5 ormore of the above miRNAs. Methods for inducing the reprogramming ofsomatic cells that involve the administration of HES-oligonucleotidescontaining expression constructs encoding an miRNA, miRNA mimic or miRNAinhibitor of 1, 2, 3, 4, 5 or more of the above miRNAs are alsoencompassed by the invention.

Methods for inducing the reprogramming of somatic cells that involve theadministration of HES-oligonucleotides containing expression constructsencoding an miRNA, miRNA mimic or miRNA inhibitor of 1, 2, 3, 4, 5 ormore of the above miRNAs are also encompassed by the invention.“Expression construct” means any double-stranded DNA or double-strandedRNA designed to transcribe an RNA of interest, e.g., a construct thatcontains at least one promoter which is or may be operably linked to adownstream gene, coding region, or polynucleotide sequence of interest(e.g., a cDNA or genomic DNA fragment that encodes a polypeptide orprotein, or an RNA effector molecule, e.g., an antisense RNA,triplex-forming RNA, ribozyme, an artificially selected high affinityRNA ligand (aptamer), a double-stranded RNA, e.g., an RNA moleculecomprising a stem-loop or hairpin dsRNA, or a bi-finger or multi-fingerdsRNA or a microRNA, or any RNA of interest). An “expression construct”includes a double-stranded DNA or RNA comprising one or more promoters,wherein one or more of the promoters is not in fact operably linked to apolynucleotide sequence to be transcribed, but instead is designed forefficient insertion of an operably-linked polynucleotide sequence to betranscribed by the promoter. Transfection or transformation of theexpression construct into a recipient cell allows the cell to express anRNA effector molecule, polypeptide, or protein encoded by the expressionconstruct. An expression construct may be a genetically engineeredplasmid, virus, recombinant virus, or an artificial chromosome derivedfrom, for example, a bacteriophage, adenovirus, adeno-associated virus,retrovirus, lentivirus, poxvirus, or herpesvirus, etc. An expressionconstruct can be replicated in a living cell, or it can be madesynthetically.

In particular embodiment, the HES-oligonucleotides contain or encodetandem copies of an miRNA, miRNA mimic and or miRNA inhibitor. Forexample, in some embodiments, the HES-oligonucleotide contains anexpression construct that encodes one or more tandem copies of one ormore miRNAs, miRNA mimics and/or miRNA inhibitors wherein the codedsequences are expressed in cis or trans from a single transcription unitor multiple polycistronic transcription units to generate a plurality(e.g., 2, 3, 4, or more) of the same or different, miRNAs, miRNA mimicsand/or miRNA inhibitors within the cell (see, e.g., Chung et al. (2006)Nucleic Acids Research 34:7, U.S. Pat. No. 6,471,957, and U.S. Publ.Nos. US 2006/0228800 and US 2011/0105593, the contents of each of whichis hereby incorporated by reference in its entirety.

Somatic cells that can be reprogramed according to the methods of theinvention can be obtained from any source using techniques known tothose of skill in the art, including from a subject to which thereprogramed cells are optionally readministered. Examples of human andmouse sources of somatic cells that can be used according to the methodsof the invention, include, but are not limited to human foreskinfibroblasts, human dermal fibroblasts (HDFs), human adipose stromalcells (hASCs), various human cancer cell lines, mouse embryonicfibroblasts (MEFs), and mouse adipose stromal cells (mASCs).

In some embodiments, the methods of the invention involve the step ofinducing the somatic reprogramed cells to differentiate into aprogenitor or terminal cell lineage by administering to the cells one ormore HES-oligonucleotides containing or encoding a miRNA, miRNA mimic ormiRNA inhibitor that drives cell lineage specification, for example, tohematopietic cells, cardiomyocytes, hepatocytes, or neurons.

The ability of the HES-oligonucleotides of the invention to safely andefficiently delivery cell nuclear reprogramming oligonucleotides such ascertain miRNAs and miRNAs into somatic cell populations additionallymakes the methods of the invention amenable to a large-scalehigh-throughput generation of patient-specific iPSC-like cells fromlarge patient populations for therapeutic uses, that to date, has beenhampered by the low reprogramming efficiency and cell cytoxicityconcerns presented by conventional nucleic acid delivery systems.

Exemplary Therapeutic Applications of HES-Oligonucleotides

As will be immediately apparent to a person of skill in the art, due inpart to the surprising highly efficient in vivo delivery ofoligonucleotides into cells, the HES-oligonucleotide complexes of theinvention essentially have limitless applications in modulating targetnucleic acid and protein levels and activity and are particularly usefulin therapeutic applications.

Non limiting examples of diseases and disorder that may be treated withthe HES-oligonucleotides of the invention include, a proliferativedisorder (e.g., a cancer, such as hematological cancers (e.g., AML, CML,CLL and multiple myeloma) and solid tumors (e.g., melanoma, renalcancer, pancreatic cancer, prostate cancer, ovarian cancer, breastcancer, NSCLC), immune (e.g., ulcerative colitis, Crohn's disease, IBD,psoriasis, asthma, autoimmune diseases such as rheumatoid arthritis,multiple sclerosis, and SLE) and inflammatory diseases, neurologicdiseases (e.g., diabetic retinopathy, Duchenne's muscular dystrophy,myotinic dystrophy, Huntington's disease and spinal muscular atrophy andother neurodegenerative diseases), metabolic diseases (e.g., type IIdiabetes, obesity), cardiovascular diseases (e.g., clotting disorders,thrombosis, coronary artery disease, restenosis, amyloidosis,hemophilia, anemia, hemoglobulinopathies, atherosclerosis, highcholesterol, high tryglycerides), endocrine related diseases anddisorders (e.g., NASH, diabetes mellitus, diabetes insipidus, Addison'sdisease, Turner syndrome, Cushing's syndrome, osteoporosis,) andinfectious disease. Thus, in one embodiment, the invention provides amethod of treating a disease in a subject comprising administering to asubject that has been diagnosed with the disease, a therapeuticallyeffective amount of an HES-oligonucleotide containing a therapeuticoligonucleotide specifically hybridizes to a nucleic acid associatedwith the disease or disorder or a symptom thereof.

In additional embodiments, the disease or disorder treated with anHES-oligonucleotide of the invention is a disease or disorder of thekidneys, liver, lymph nodes, spleen or adipose tissue.

The invention also provides a method of monitoring the delivery of atherapeutic oligonucleotide to a cell or tissue in a subject, comprisingadministering to the subject an HES-oligonucleotide complex containing atherapeutic oligonucleotide and monitoring the fluorescence of cells ortissue in the subject, wherein an increased fluorescence in the cells ortissue of the subject indicates that the therapeutic oligonucleotide hasbeen delivered to the cells or tissue of the subject.

In particular embodiments, the invention provides a method of monitoringthe delivery of a therapeutic oligonucleotide to a cell or tissue in asubject, comprising administering to the subject an HES-oligonucleotidecomplex containing a therapeutic oligonucleotide and monitoring thefluorescence of cells or tissue in the subject, wherein an increasedfluorescence in the cells or tissue of the subject to a predeterminedvalue indicates that a therapeutically effective amount of theoligonucleotide has been delivered to the cells or tissue of thesubject. In particular embodiments, the predetermined value isdetermined by extrapolating from corresponding changes in fluorescenceassociated with delivery of a therapeutically effective amount of thetherapeutic HES-oligonucleotide to cells in vitro or throughquantitative fluorescence modeling analysis.

The invention also encompasses a method of treating a disease ordisorder characterized by the under expression of a nucleic acid in asubject, comprising administering to the subject an HES-oligonucleotidecomplex containing an oligonucleotide which comprises or encodes thenucleic acid or increases the endogenous expression, processing orfunction of the nucleic acid (e.g., by binding regulatory sequences inthe gene encoding the nucleic acid) and which acts to increase the levelof the nucleic acid and/or increase its function in the cell. In someembodiments, the oligonucleotide comprises a sequence substantially thesame as a nucleic acid comprising or encoding the nucleic acid.

The invention also encompasses a method of treating a disease ordisorder characterized by the underexpression of a protein in a subject,comprising administering to the subject an HES-oligonucleotide complex,containing an oligonucleotide which encodes the protein or increases theendogenous expression, processing or function of the protein in thesubject.

In another embodiment, the invention provides a method of treatingcancer or one or more conditions associated with cancer by administeringa therapeutically effective amount of an HES-oligonucleotide to asubject in need thereof. “Cancer,” “tumor,” or “malignancy” are usedherein as synonymous terms and refer to any of a number of diseases thatare characterized by uncontrolled, abnormal proliferation of cells, theability of affected cells to spread locally or through the bloodstreamand lymphatic system to other parts of the body (metastasize), as wellas any of a number of known characteristic structural and/or molecularfeatures. A “cancerous tumor” or “malignant cell” is understood as acell having specific structural properties, lacking differentiation andbeing capable of invasion and metastasis. Examples of cancers that maybe treated using HES-oligonucleotide complexes of the invention includesolid tumors and hematologic cancers. Additional, examples of cancersthat can be treated using HES-oligonucleotide complexes of the inventioninclude, breast, lung, brain, bone, liver, kidney, colon, head and neck,ovarian, hematopoietic (e.g., leukemia), and prostate cancer. Furtherexamples of cancer that can be treated using HES-oligonucleotidecomplexes include, but are not limited to, carcinoma, lymphoma, myeloma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include, but are not limited to, squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma and various types of head and neck cancers.

In additional embodiments, a therapeutically effective amount of anHES-oligonucleotide is administered to treat a hematologic cancer. Infurther embodiments, the, HES-oligonucleotide is administered to treat acancer selected from: lymphoma, leukemia, myeloma, lymphoid malignancy,cancer of the spleen, and cancer of the lymph nodes. In additionalembodiments, a therapeutically effective amount of anHES-oligonucleotide complex is administered to treat a lymphoma selectedfrom: Burkitt's lymphoma, diffuse large cell lymphoma, follicularlymphoma, Hodgkin's lymphoma, mantle cell lymphoma, marginal zonelymphoma, mucosa-associated-lymphoid tissue B cell lymphoma,non-Hodgkin's lymphoma, small lymphocytic lymphoma, and a T celllymphoma. In additional embodiments, a therapeutically effective amountof an HES-oligonucleotide complex is administered to treat a leukemiaselected from: chronic lymphocytic leukemia, B cell leukemia (CD5+ Blymphocytes), chronic myeloid leukemia, lymphoid leukemia, acutelymphoblastic leukemia, myelodysplasia, myeloid leukemia, acute myeloidleukemia, and secondary leukemia. In additional embodiments, atherapeutically effective amount of an HES-oligonucleotide complex isadministered to treat multiple myeloma. Other types of cancer and tumorsthat can be treated using HES-oligonucleotides are described herein orotherwise known in the art.

In particular embodiments, the HES-oligonucleotide contains anoligonucleotide selected from: AVI-4557 (Cyp 3A4m; AVI Biopharma),ISIS-2372 (Survivin; ISIS); Gem-640 (XIAP; Hybridon), Atu027 (PKN3;Silence Therapeutics), CEQ508 (B catenin; Marina Biotech), GEM 231 (PKAR1α subunit; Idera), Affinitak (Aprinocarsen, ISIS 3521/LY900003; PKC-α;ISIS/Lilly); Aezea (OL(1)p53/EL-625; p53; Eleos Pharma); ISIS 2503(H-ras; ISIS), EZN-2968 (Hif-1α; Enzon Pharmaceuticals); G4460/LR 3001

(c-Myb; Inex/Genta); LErafAON (c-Raf; NeoPharm), ISIS 5132 (c-Raf;ISIS), Genasense (Oblimersen/G3139; Bcl-2; Genta); SPC2996 (Bcl-2;Santaris Pharma), OGX-427 (Hsp27; ISIS/OncoGene X), LY2181308 (Surivin;Lilly), LY2275796 (EIF4E; Lilly), ISIS-STAT3 Rx (STATS; ISIS), OGX-011(Custirsen; clusterin; Teva), Veglin (VEGF; VasGene Therapeutics,AP12009 (TGF-β2; Antisense Pharma), GTI-2501

(Ribonucleotide Reductase R1; Lorus Therapeutics), Gem-220 (VEGF;Hybridon); Gem-240 (MEM2; Hybridon), CALAA-19 (M2 subunit ribonucleotidereductase; Arrowhead Research Corporation), Trabedersen (AP 12009;TGFB2; Antisense), GTI-2040 (Ribonucleotide Reductase R2 LorusTherapeutics), AEG 35156 (XIAP; Aegera Pharma), and MG 98 (DNAmethyltransferase; MethylGene/MGI Pharma/British Biotech). In particularembodiments, an oligonucleotide in an HES-oligonucleotide of theinvention competes for target binding with one of the aboveoligonucleotides.

In additional embodiments, the HES-oligonucleotide contains anoligonucleotide that specifically hybridizes to a nucleic acid sequencethat modulates apoptosis, cell survival, angiogenesis, metastasis,aberrant gene regulation, cell cycle, mitogenic pathways and/or growthsignaling. In further embodiments, the HES-oligonucleotide contains anoligonucleotide that specifically hybridizes to a nucleic acid sequencethat modulates the expression of a protein selected from: from: EGFR,HER-2/neu, ErbB3, cMet, p561ck, PDGFR, VEGF, VEGFR, FGF, FGFR, ANG1,ANG2, bFGF, TIE2, protein kinase C-alpha (PKC-alpha), p561ck PKA,TGF-beta, IGFIR, P12, MDM2, BRCA, IGF1, HGF, PDGF, IGFBP2, IGFIR, HIF1alpha, ferritin, transferrin receptor, TMPRSS2, IRE, HSP27, HSP70,HSP90, MITF, clusterin, PARP1C-fos, C-myc, n-myc, C-raf, B-raf, A1,H-raf, Skp2, K-ras, N-ras, H-ras, farensyltransferase, c-Src, Jun, Fos,Bcr-Abl, c-Kit, EphA2, PDGFB, ARF, NOX1, NF1STAT3, E6/E7, APC, WNT, betacatenin, GSK3b, PI3k, mTOR, Akt, PDK-1, CDK, Mek1, ERK1, AP-1, p53, Rb,Syk, osteopontin, CD44, MEK, MAPK, NF kappa beta, E cadherin, cyclin D,cyclin E, Bcl-2, Bax, BXL-XL, BCL-W, MCL1, ER, MDR, telomerase,telomerase reverse transcriptase, a DNA methyltransferase, a histonedeacetlyase (e.g., HDAC1 and HDAC2), an integrin, an IAP, an aurorakinase, a metalloprotease (e.g., MMP2, MMP3 and MMP9), a proteasome, anda metallothionein gene.

In another embodiment, the invention provides a method of treatingcancer or one or more conditions associated with cancer by administeringan HES-oligonucleotide in combination with one or more therapiescurrently being used, have been used, or are known to be useful in thetreatment of cancer or conditions associated with cancer.

In some embodiments, the invention provides a method of treating aninflammatory or other disease or disorder of the immune system, or oneor more conditions associated with an inflammatory or other disease ordisorder of the immune system, said method comprising administering to asubject in need thereof (i.e., having or at risk of having aninflammatory or other immune system disease or disorder), atherapeutically effective amount of one or more HES-oligonucleotides ofthe invention. As immediately apparent to those skilled in the art, anytype of immune or inflammatory disease or condition resulting from orassociated with an immune system or inflammatory disease can be treatedin accordance with the methods of the invention. In particularembodiments, the invention is directed to treating an immune systemand/or inflammatory disease or disorder, or one or more conditionsassociated with such an immune disease or disorder.

The term “inflammatory disorders”, as used herein, refers to thosediseases or conditions that are characterized by one or more of thesigns of pain (dolor, from the generation of noxious substances and thestimulation of nerves), heat (calor, from vasodilatation), redness(rubor, from vasodilatation and increased blood flow), swelling (tumor,from excessive inflow or restricted outflow of fluid), and loss offunction (functio laesa, which may be partial or complete, temporary orpermanent). Inflammation takes many forms and includes, but is notlimited to, inflammation that is one or more of the following: acute,adhesive, atrophic, catarrhal, chronic, cirrhotic, diffuse,disseminated, exudative, fibrinous, fibrosing, focal, granulomatous,hyperplastic, hypertrophic, interstitial, metastatic, necrotic,obliterative, parenchymatous, plastic, productive, proliferous,pseudomembranous, purulent, sclerosing, seroplastic, serous, simple,specific, subacute, suppurative, toxic, traumatic, and/or ulcerative.Inflammatory disorders additionally include but are not limited to thoseaffecting the blood vessels (polyarteritis, temporarl arteritis); joints(arthritis: crystalline, osteo-, psoriatic, reactive, rheumatoid,Reiter's); gastrointestinal tract (Disease); skin (dermatitis); ormultiple organs and tissues (systemic lupus erythematosus). The terms“fibrosis” or “fibrosing disorder,” as used herein, refers to conditionsthat follow acute or chronic inflammation and are associated with theabnormal accumulation of cells and/or collagen and include but are notlimited to fibrosis of individual organs or tissues such as the heart,kidney, joints, lung, or skin, and includes such disorders as idiopathicpulmonary fibrosis and cryptogenic fibrosing alveolitis. In particularembodiments, the inflammatory disorder is selected from the groupconsisting of asthma, allergic disorders, and rheumatoid arthritis.

In further embodiment, the disorder or disorder of the immune system isan autoimmune disease. Autoimmune diseases, disorders or conditions thatmay be treated using the HES-oligonucleotide complexes of the inventioninclude, but are not limited to, autoimmune hemolytic anemia, autoimmuneneonatal thrombocytopenia, idiopathic thrombocytopenia purpura,autoimmune neutropenia, autoimmunocytopenia, hemolytic anemia,antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy,allergic encephalomyelitis, myocarditis, relapsing polychondritis,rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy),Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies,Purpura (e.g., Henloch Scoenlein purpura), Reiter's Disease, Stiff-ManSyndrome, Autoimmune Pulmonary Inflammation, myocarditis, IgAglomerulonephritis, dense deposit disease, rheumatic heart disease,Guillain-Barre Syndrome, insulin dependent diabetes mellitus, andautoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism(i.e., Hashimoto's thyroiditis, systemic lupus erythematous, discoidlupus, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities forexample, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulinresistance, autoimmune hemolytic anemia, autoimmune thrombocytopenicpurpura, rheumatoid arthritis, scleroderma with anti-collagenantibodies, mixed connective tissue disease,polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison'sdisease, infertility, glomerulonephritis such as primaryglomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren'ssyndrome, diabetes mellitus, and adrenergic drag resistance (includingadrenergic drug resistance with asthma or cystic fibrosis), chronicactive hepatitis, primary biliary cirrhosis, other endocrine glandfailure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria,atopic dermatitis, asthma, inflammatory myopathies, and otherinflammatory, granulomatous, degenerative, and atrophic disorders. Inparticular embodiments, the autoimmune disease or disorder is selectedfrom Crohn's disease, Systemic lupus erythematous (SLE), inflammatorybowel disease, psoriasis, diabetes, ulcerative colitis, multiplesclerosis, and rheumatoid arthritis.

In some embodiment, the invention is directed to methods of treating animmune or cardiovascular disease comprising administering to a subject atherapeutically effective amount of an HES-oligonucleotide. Inparticular embodiments, the HES-oligonucleotide complex contains anoligonucleotide selected from: Alicaforsen (ICAM-1; ISIS 2302), QPI-1002(p53; Silence Thera/Novartis/Quark), XEN701 (Isis/XenonPharmaceuticals), ISIS 104838 (TNF-α; ISIS/Orasense), EPI-2010 (RASON;Adenosine A1 receptor; Epigenesis/Genta), Plazomicin (Isis/Achaogen),ALN-PCS02 (PCSK9; Alnylam), ALN-AT3 (SERPINC1; Alnylam), ALN-HPN (TFR2;Alnylam), ALN-HPN (TMPRSS6; Alnylam), ASM8-003 (CCR3; TopigenPharmaceuticals), ISIS CRP Rx (CRP; ISIS), Kynamro™ (ISIS 301012;Apo-B100; ISIS/Genzyme), ISIS-APOCIII Rx (ApoCIII; ISIS), ISIS-APO(a)(Apo(a); ISIS); ISIS-FVII rx Factor VII; ISIS), and ISIS-FXI (Factor XI;ISIS). In particular embodiments, an oligonucleotide in anHES-oligonucleotide complex of the invention competes with one of theabove oligonucleotides for target binding.

In some embodiments, the invention provides a method of treating aninfectious disease or one or more conditions associated with aninfectious disease, said method comprising administering to a subject inneed thereof (i.e., having or at risk of having an infectious disease),a therapeutically effective amount of one or more HES-oligonucleotidesof the invention. In some embodiments the infectious disease is a viralinfection, a bacterial infection, a fungal infection or a parasiteinfection.

In some embodiments, the invention provides a method of treating aninfection or condition associated with a category A infectious agent ordisease, said method comprising administering to a subject in needthereof (i.e., having or at risk of having an infectious disease), atherapeutically effective amount of one or more HES-oligonucleotides ofthe invention. In particular embodiments, the infectious agent isselected from Bacillus anthracis, Clostridium botulinum toxin, yersinapestis, variola major a filovirus (e.g., Ebola and Marburg) and anarenavirus (e.g., Lassa and Machupo). In particular embodiments, thecondition treated according to the methods of the invention is selectedfrom: anthrax, botulism, plague, smallpox, tularemia, and a viralhemorrhagic fever.

In some embodiments, the invention provides a method of treating aninfection or condition associated with a category B infectious agent ordisease, said method comprising administering to a subject in needthereof (i.e., having or at risk of having an infectious disease), atherapeutically effective amount of one or more HES-oligonucleotides ofthe invention. In particular embodiments, the infectious agent isselected from: a Bacilla species, Clostridium perfringens, a Salmonellaspecies, E. coli 0157:H7, Shigella, Burkholderia pseudomallei, Chyamydiapsittaci, Coxiella burnetii, Rickettsia prowazekii, a viral encephalitisalphavirus (e.g., Venezuelan equine encephalitis, eastern equineencephalitis, western equine encephalitis), Vibrio cholerae andCryptosporidium parvum. In particular embodiments, the condition treatedaccording to the methods of the invention is selected from: Brucellosis,epsilon toxin of Clostridium perfringens, food poisoning, Glanders,Melioidosis, Psittacosis, Q fever, ricin toxin poisoning, typhus fever,viral encephalitis and dysentery.

In some embodiments, the invention provides a method of treating a viralinfection or one or more conditions associated with a viral infection,said method comprising administering to a subject in need thereof (i.e.,having or at risk of having a viral infection), a therapeuticallyeffective amount of one or more HES-oligonucleotides of the invention.As immediately apparent to those skilled in the art, any type of viralinfection or condition resulting from or associated with a viralinfection (e.g., a respiratory condition) can be treated in accordancewith the methods of the invention. In particular embodiments, the viraldisease or disorder is an infection or condition associated with amember selected from: Ebola, Marburg, Junin, Denge West Nile, Lassa SARSCo-V, Japanese encephalitis, Venezuelan equine encephalitis, Saint Louisencephalitis, Manchupo, Yellow fever, and Influenza.

Examples of viruses which cause viral infections and conditions that canbe treated with the HES-oligonucleotides of the invention include, butare not limited to, infections and conditions associated withretroviruses (e.g., human T-cell lymphotrophic virus (HTLV) types I andII and human immunodeficiency virus (HIV)), herpes viruses (e.g., herpessimplex virus (HSV) types I and II, Epstein-Barr virus, HHV6-HHV8, andcytomegalovirus), arenavirus (e.g., lassa fever virus), paramyxoviruses(e.g., morbillivirus virus, human respiratory syncytial virus, mumps,hMPV, and pneumovirus), adenoviruses, bunyaviruses (e.g., hantavirus),cornaviruses, filoviruses (e.g., Ebola virus), flaviviruses (e.g.,hepatitis C virus (HCV), yellow fever virus, and Japanese encephalitisvirus), hepadnaviruses (e.g., hepatitis B viruses (HBV)),orthomyoviruses (e.g., influenza viruses A, B and C and PIV),papovaviruses (e.g., papillomavirues), picornaviruses (e.g.,rhinoviruses, enteroviruses and hepatitis A viruses), poxviruses,reoviruses (e.g., rotavirues), togaviruses (e.g., rubella virus), andrhabdoviruses (e.g., rabies virus).

In additional embodiments, the invention provides a method of treatingor alleviating conditions associated with viral respiratory infectionsassociated with or that cause the common cold, viral pharyngitis, virallaryngitis, viral croup, viral bronchitis, influenza, parainfluenzaviral diseases (“PIV”) diseases (e.g., croup, bronchiolitis, bronchitis,pneumonia), respiratory syncytial virus (“RSV”) diseases,metapneumavirus diseases, and adenovirus diseases (e.g., febrilerespiratory disease, croup, bronchitis, and pneumonia).

In some embodiment, the HES-oligonucleotide contains an oligonucleotideselected from: AVI-4065 (HCV; AVI Biopharma), VRX496 (HIV; VIRxSYScorporation), Miravirsen (antimiR-122, Santaris), GEM 91(Trecorvirsen)/92; Gag HIV; Hybridon), Vitravene (Fomivirsen; CMV;ISIS/Novartis), ALN-RSV01 (RSV; Alnylam), AVI-6002 (Ebola; AVIBiopharma), AVI-6003 (Ebola; AVI Biopharma), MBI-1121 (humanpapillomavirus; Hybridon), ARC-520 (HPV hepatitis; Arrowhead ResearchCorporation) and AVI-6001 (Influenza/avian flu; AVI Biopharma). Inparticular embodiments, an oligonucleotide in an HES-oligonucleotide ofthe invention competes for target binding with one of the aboveoligonucleotides.

In an additional embodiment, the invention provides a method of treatinga viral infection or one or more conditions associated with a viralinfection by administering a combination of at least 1, at least 2, atleast 3, at least 4, or at least 5 HES-oligonucleotides of theinvention. In some embodiments at least 2, at least 3, or at least 4 ofthe HES-oligonucleotides specifically hybridizes to the same targetnucleic acid. In additional embodiments, at least 2, at least 3, or atleast 4 or at least 5 of the HES-oligonucleotides bind to a differenttarget nucleic acid.

In one embodiment, the invention provides a method of treating afilovirus (e.g., Ebola and Marbury) infection or one or more conditionsassociated with the infection by administering to a patient in needthereof, a therapeutically effective amount of HES-oligonucleotides thatspecifically hybridize to at least 1, at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9 or atleast 10 RNA sequences of a filovirus. In particular embodiments, theHES-oligonucleotides bind VP35, VP24 and/or RNA polymerase L. In furtherembodiments one or more of the HES-oligonucleotides are PMOs or PPMOs.In additional embodiments one or more of the HES-oligonucleotides is anantisense, an siRNA or an shRNA.

In one embodiment, the invention provides a method of treating an Ebolavirus infection or one or more conditions associated with the infectionby administering to a patient in need thereof, HES-oligonucleotides thatbind to at least 1, at least 2, at least 3, at least 4, at least 5, at,least 6, at least 7, at least 8, at least 9 or at least 10 Ebola RNAsequences. In particular embodiments, the HES-oligonucleotides bindVP24, VP35, and/or RNA polymerase L. In additional embodiments, theHES-oligonucleotides bind VP24, VP30, VP35, VP40, NP, GP and/or RNApolymerase L. In further embodiments one or more of theHES-oligonucleotides are PMOs or PPMOs. In additional embodiments one ormore of the HES-oligonucleotides is an antisense, an siRNA or an shRNA.

In one embodiment, the invention provides a method of treating anFlaviviridae (e.g., West Nile, yellow fever, Japanese encephalitis, anddengue viruses) viral infection or one or more conditions associatedwith the infection by administering to a patient in need thereof, atherapeutically effective amount of HES-oligonucleotides thatspecifically hybridize to at least 1, at least 2, at least 3, at least4, or at least 5 RNA sequences of a member of the family Flaviviridae.In particular embodiments, the HES-oligonucleotides bind the highlyconserved non coding sequence in the 5′ or 3′ regions of the viralgenome, or sequence corresponding to the envelope coding gene (E). Infarther embodiments one or more of the HES-oligonucleotides are PMOs orPPMOs. In additional embodiments one or more of the HES-oligonucleotidesis an antisense, an siRNA or an shRNA.

In one embodiment, the invention provides a method of treating anArenavirideae (e.g., Lassa, Junin and Machupo viruses) family viralinfection or one or more conditions associated with the infection byadministering to a patient in need thereof, a therapeutically effectiveamount of HES-oligonucleotides that specifically hybridizes to at least1, at least 2, at least 3, at least 4, or at least 5 RNA sequences of amember of the family Arenavirideae. In particular embodiments, theHES-oligonucleotides bind the highly conserved non coding sequence inthe 5′ or 3′ viral mRNAs transcript coding for the Z protein(zinc-binding protein), L protein (viral polymerase), or the GPC(glycoprotein precursor) protein. In further embodiments one or more ofthe HES-oligonucleotides are PMOs or PPMOs. In additional embodimentsone or more of the HES-oligonucleotides is an antisense, an siRNA or anshRNA.

In one embodiment, the invention provides a method of treating aSARS-associated coronavirus (SARS Co-V) infection or one or moreconditions associated with the infection by administering to a patientin need thereof, a therapeutically effective amount ofHES-oligonucleotides that specifically hybridize to at least 1, at least2, at least 3, at least 4, or at least 5 family SARS Co-V nucleic acidsequences. In particular embodiments, the HES-oligonucleotides bind thereplica se gene (orf 1a/1b), orf 1b ribosomal frameshift point, 5′untranslated region (UTR) of the transcription regulatory sequence(TRS), 3′ UTR of the TRS sequence, spike protein-coding region and/orthe NSP12 region. In further embodiments one or more of theHES-oligonucleotides are PMOs or PPMOs. In additional embodiments one ormore of the HES-oligonucleotides is an antisense, an siRNA or an shRNA.

In one embodiment, the invention provides a method of treating anRetroviridae (e.g., HIV viruses) family viral infection or one or moreconditions associated with the infection by administering to a patientin need thereof, a therapeutically effective amount ofHES-oligonucleotides that specifically hybridize to at least 2, at least3, at least 4, or at least 5 RNA sequences of a member of the familyRetroviridae. In particular embodiments, the HES-oligonucleotide(s) bindthe highly conserved regions of the gag, pol, int, and Vpu regions. Infurther embodiments one or more of the HES-oligonucleotides are PMOs orPPMOs. In additional embodiments one or more of the HES-oligonucleotidesis an antisense, an siRNA or an shRNA.

In another embodiment, the invention provides a method of treating aninfluenza A (e.g., H1N1, H3N2 and H5N1) infection or one or moreconditions associated with influenza by administering to a patient inneed thereof, a therapeutically effective amount of HES-oligonucleotidesthat specifically hybridize to at least 2, at least 3, at least 4, or atleast 5 influenza RNA sequences. In particular embodiments, theHES-oligonucleotides bind NP and PA nucleic acid sequence of the virus.In particular embodiments, the HES-oligonucleotides bind an NP, M2,and/or PB2 (e.g., targeting the AUG start codon of PA, PB1, PB2, andNP), or terminal region of NP), NS1 and/or PA nucleic acid sequence ofthe virus. In further embodiments one or more of theHES-oligonucleotides are PMOs or PPMOs. In additional embodiments one ormore of the HES-oligonucleotides is an antisense, an siRNA or an shRNA.

In an additional embodiment, the invention provides a method of treatingan alphavirus (equine encephalitis virus (VEEV)) infection or one ormore conditions associated with an alphavirus infection by administeringto a patient in need thereof, a therapeutically effective amount ofHES-oligonucleotides that specifically hybridize to at least 2, at least3, at least 4, or at least 5 alphavirus RNA sequences. In particularembodiments, the HES-oligonucleotides bind NP and PA nucleic acidsequence of the virus. In particular embodiments, theHES-oligonucleotides bind an nsp1, nsp4 and/or E1 RNA sequence of thevirus. In further embodiments one or more of the HES-oligonucleotidesare PMOs or PPMOs. In additional embodiments one or more of theHES-oligonucleotides is an antisense, an siRNA or an shRNA.

In some embodiments, the invention provides a method of treating abacterial infection or one or more conditions associated with abacterial infection, said method comprising administering to a subjectin need thereof (i.e., having or at risk of having a bacterialinfection), a therapeutically effective amount of one or moreHES-oligonucleotides of the invention. Any type of bacterial infectionor condition resulting from, or associated with a bacterial infectioncan be treated using the compositions and methods of the invention. Inparticular embodiments, the bacterial infection or condition treatedaccording to the methods of the invention is associated with a member ofa bacterial genus selected from: Salmonella, Shigella, Chlamydia,Helicobacter, Yersinia, Bordatella, Pseudomonas, Neisseria, Vibrio,Haemophilus, Mycoplasma, Streptomyces, Treponema, Coxiella, Ehrlichia,Brucella, Streptobacillus, Fusospirocheta, Spirillum, Ureaplasma,Spirochaeta, Mycoplasma, Actinomycetes, Borrelia, Bacteroides,Trichomoras, Branhamella, Pasteurella, Clostridium, Corynebacterium,Listeria, Bacillus, Erysipelothrix, Rhodococcus, Escherichia,Klebsiella, Pseudomanas, Enterobacter, Serratia, Staphylococcus,Streptococcus, Legionella, Mycobacterium, Proteus, Campylobacter,Enterococcus, Acinetobacter, Morganella, Moraxella, Citrobacter,Rickettsia and Rochlimeae. In further embodiments, the bacterialinfection or condition treated according to the methods of the inventionis associated with a member of a bacterial genus selected from: P.aeruginosa; E, coli, P. cepacia, S. epidermis, E. faecalis, S.pneumonias, S. aureus, N. meningitidis, S. pyogenes, Pasteurellamultocida, Treponema pallidum, and P. mirabilis. In some embodiments,the bacterial infection is an intracellular bacterial infection. Inadditional embodiments, the invention provides a method of treating anbacterial infection or one or more conditions associated with abacterial infection by administering to a patient in need thereof, atherapeutically effective amount of HES-oligonucleotides thatspecifically hybridize to at least 1, at least 2, at least 3, at least4, or at least 5 nucleic acid sequences of at least 1, at least 2, atleast 3, at least 4, or at least 5 of the above bacteria.

In additional embodiments, the invention provides a method of treating afungal infection or one or more conditions associated with a fungalinfection, said method comprising administering to a subject in needthereof (i.e., having or at risk of having a fungal infection), atherapeutically effective amount of one or more HES-oligonucleotides ofthe invention. Any type of fungal infection or condition resulting fromor associated with a fungal infection can be treated using thecompositions and methods of the invention. In particular embodiments,the fungal infection or condition treated according to the methods ofthe invention is associated with a fungus selected from: Cryptococcusneoformans; Blastomyces dermatitidis; Aiellomyces dermatitidis;Histoplasma capsulatum; Coccidioides immitis; a Candida species,including C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondiiand C. krusei, an Aspergillus species, including A. fumigatus, A. flavusand A. niger; a Rhizopus species; a Rhizomucor species; aCunninghammella species; a Apophysomyces species, including A.saksenaea, A. mucor and A. absidia; Sporothrix schenckii,Paracoccidioides brasiliensis; Pseudalleseheria boydii, Torulopsisglabrata; a Trichophyton species, a Microsporum species and aDermatophyres species, or any other fungus (e.g., yeast) known oridentified to be pathogenic. In additional embodiments, the inventionprovides a method of treating a fungal infection or condition associatedwith a fungi infection by administering to a patient in need thereof, atherapeutically effective amount of HES-oligonucleotides thatspecifically hybridize to at least 1, at least 2, at least 3, at least4, or at least 5 nucleic acid sequences of at least 1, at least 2, atleast 3, at least 4, or at least 5 of the above funghi.

In additional embodiments, the invention provides a method of treating aparasite infection or one or more conditions associated with a parasiteinfection, said method comprising administering to a subject in needthereof (i.e., having or at risk of having a parasite infection), atherapeutically effective amount of one or more HES-oligoliucleotides ofthe invention. Any type of parasite infection or condition resultingfrom or associated with a parasite infection can be treated using thecompositions and methods of the invention. In particular embodiments,the parasite infection or condition treated according to the methods ofthe invention is associated with a parasite selected from: a member ofthe Apicomplexa phylum such as, Babesia, Toxoplasma, Plasmodium,Eimeria, Isospora, Atoxoplasma, Cystoisospora, Hammondia, Besniotia,Sarcocystis, Frenkelia, Haemoproteus, Leucocytozoon, Theileria,Perkinsus or Gregarina spp.; Pneumocystis carinii; a member of theMicrospora phylum such as, Nosema, Enterocytozoon, Encephalitozoon,Septata, Mrazekia, Amblyospora, Arneson, Glugea, Pleistophora andMicrosporidium spp.; and a member of the Ascetospora phylum such as,Haplosporidium spp. In further embodiments, the parasite infection orcondition treated according to the methods of the invention isassociated with a parasite species selected from: Plasmodium falciparum,P. vivax, P. ovale, P. malaria; Toxoplasma gondii; Leishmania mexicana,L. tropica, L. major, L. aethiopica, L. donovani, Trypanosoma cruzi, T.brucei, Schistosoma mansoni, S. haematobium, S. japonium, Trichinellaspiralis; Wuchereria bancrofti; Brugia malayli; Entamoeba histolytica;Enterobius vermiculoarus; Taenia solium, T. saginata, Trichomonasvaginatis, T. hominis, T. tenax; Giardia lamblia; Cryptosporidiumparvum; Pneumocytis carinii, Babesia Bovis, B. divergens, B. microti,Isospora belli, L. hominis; Dientamoeba fragilis; Onchocerca volvulus;Ascaris lumbricoides; Necator americanis; Ancylostoma duodenale;Strongyloides stercoralis; Capillaria philippinensis; Angiostrongyluscantonensis; Hymenolepis nana; Diphyllobothrium latum; Echinococcusgranulosus, E. multilocularis; Paragonimus westermani, P. caliensis;Chlonorchis sinensis; Opisthorchis felineas, G. Viverini, Fasciolahepatica, Sarcoptes scabiei, Pediculus humanus; Phthirlus pubis; andDermatobia hominis, as well as any other parasite known or identified tobe pathogenic. In additional embodiments, the invention provides amethod of treating an parasite infection or one or more conditionsassociated with a parasite infection by administering to a patient inneed thereof, a therapeutically effective amount of HES-oligonucleotidesthat specifically hybridize to at least 1, at least 2, at least 3, atleast 4, or at least 5 nucleic acid sequences of at least 1, at least 2,at least 3, at least 4, or at least 5 of the above parasites.

In another embodiment, the invention provides a method of treating aviral infection or one or more conditions associated with a viralinfection by administering an HES-oligonucleotide of the invention incombination with one or more therapies currently being used, have beenused, or are known to be useful in the treatment of a viral infection orconditions associated with a viral infection, including but not limitedto, anti-viral agents such as amantadine, oseltamivir, ribaviran,palivizumab, and anamivir. In certain embodiments, a therapeuticallyeffective amount of one or more HES-oligonucleotides of the invention isadministered in combination with one or more anti-viral agents such as,but not limited to, amantadine, rimantadine, oseltamivir, znamivir,ribaviran, RSV-IVIG (i.e., intravenous immune globulin infusion)(RESPIGAM™), and palivizumab.

In some embodiments, the invention provides a method of treating anrespiratory disease or one or more conditions associated with arespiratory disease, said method comprising administering to a subjectin need thereof (i.e., having or at risk of having an respiratorydisease), a therapeutically effective amount of one or moreHES-oligonucleotides of the invention. The term “respiratory disease,”as used herein, refers to a disease affecting organs involved inbreathing, such as the nose, throat, larynx, trachea, bronchi, andlungs. Respiratory diseases that can be treated according to the methodsof the invention include, but are not limited to, asthma, adultrespiratory distress syndrome and allergic (extrinsic) asthma,non-allergic (intrinsic) asthma, acute severe asthma, chronic asthma,clinical asthma, nocturnal asthma, allergen-induced asthma,aspirin-sensitive asthma, exercise-induced asthma, isocapnichyperventilation, child-onset asthma, adult-onset asthma, cough-variantasthma, occupational asthma, steroid-resistant asthma, seasonal asthma,seasonal allergic rhinitis, perennial allergic rhinitis, chronicobstructive pulmonary disease, including chronic bronchitis oremphysema, pulmonary hypertension, interstitial lung fibrosis and/orairway inflammation and cystic fibrosis, and hypoxia.

In some embodiments, the invention provides a method of treating anneurological condition or disorder, said method comprising administeringto a subject in need thereof (i.e., having or at risk of having aneurological condition or disorder), a therapeutically effective amountof one or more HES-oligonucleotides of the invention. The term“neurological condition or disorder” is used herein to refer toconditions that include neurodegenerative conditions, neuronal cell ortissue injuries characterized by dysfunction of the central orperipheral nervous system or by necrosis and/or apoptosis of neuronalcells or tissue, and neuronal cell or tissue damage associated withtrophic factor deprivation. Examples of neurodegenerative diseases thatcan be treated using the HES-oligonucleotide of the invention include,but are not limited to, familial and sporadic amyotrophic lateralsclerosis (FALS and ALS, respectively), familial and sporadicParkinson's disease, Huntington's disease (Huntington's chorea),familial and sporadic Alzheimer's disease, Spinal Muscular Atrophy(SMA), optical neuropathies such as glaucoma or associated diseaseinvolving retinal degeneration, diabetic neuropathy, or maculardegeneration, hearing loss due to degeneration of inner ear sensorycells or neurons, epilepsy, Bell's palsy, frontotemporal dementia withparkinsonism linked to chromosome 17 (FTDP-17), multiple sclerosis,diffuse cerebral cortical atrophy, Lewy-body dementia, Pick disease,trinucleotide repeat disease, prion disorder, and Shy-Drager syndrome.Examples of neuronal cell or tissue injuries that can be treating usingHES-oligonucleotides of the invention include, but are not limited to,acute and non-acute injury found after blunt or surgical trauma(including post-surgical cognitive dysfunction and spinal cord or brainstem injury) and ischemic conditions restricting (temporarily orpermanently) blood flow such as that associated with global and focalcerebral ischemia (stroke); incisions or cuts for instance to cerebraltissue or spinal cord; lesions or placques in neuronal tissues;deprivation of trophic factor(s) needed for growth and survival ofcells; and exposure to neurotoxins such as chemotherapeutic agents; aswell as incidental to other disease states such as chronic metabolicdiseases such as diabetes and renal dysfunction.

In some embodiment, the invention is directed to methods of treating aneurological condition or disorder comprising administering to a subjecta therapeutically effective amount of an HES-oligonucleotide. Inparticular embodiments, the HES-oligonucleotide complex contains anoligonucleotide selected from: AVI-4658 (Dystrophin (exon-skipping); AVIBiopharma), ISIS-SMN Rx (SMN; ISIS/Biogen Idec), AVI-5126 (CABG; AVIBiopharma) and ATL1102 (VLA-4 (CD49d); ISIS/Antisense Therapeutics Ltd).In particular embodiments, an oligonucleotide in an HES-oligonucleotidecomplex of the invention competes with one of the above oligonucleotidesfor target binding.

In some embodiment, the invention is directed to methods of treating ametabolic disorder comprising administering to a subject atherapeutically effective amount of an HES-oligonucleotide. Inparticular embodiments, the HES-oligonucleotide complex contains anoligonucleotide selected from: ISIS-FGFR4 (FGFR4; ISIS), ISIS-GCCR RX(GCC; ISIS), ISIS-GCGR RX (GCG; ISIS), ISIS-PTP1B (PTP1VB; ISIS),iCo-007 (c-Raf; Isis/iCo Therapeutics Inc)ISIS-DGATRX (DGAT; ISIS),PF-04523655 (DME, Silence Thera/Pfizer/Quark), ISIS-TTR Rx (TTR:ISIS/GSI); ISIS-AAT Rx (AAT: ISIS/GSK), ALN-TTRsc (Transerythrin;Alnylam), ALN-TTR01 (Transerythrin; Alnylam), and ALN-TTR02(Transerythrin; Alnylam). In particular embodiments, an oligonucleotidein an HES-oligonucleotide complex of the invention competes with one ofthe above oligonucleotides for target binding.

In some embodiment, the invention is directed to methods of treating adisease comprising administering to a subject a therapeuticallyeffective amount of an HES-oligonucleotide. In particular embodiments,the HES-oligonucleotide complex contains an oligonucleotide selectedfrom: ATL1103-GHr Rx (GHr; ISIS/Antisense Therapeutics Ltd), EXC 001(CTGF; ISIS/Excaliard), and Atu111 (PKN3; Silence Thera). In particularembodiments, an oligonucleotide in an HES-oligonucleotide complex of theinvention competes with one of the above oligonucleotides for targetbinding.

In addition to those described above, HES-oligonucleotides of theinvention have applications including but not limited to; treatingmetabolic diseases or disorders (e.g., mellitus, obesity, highcholesterol, high triglycerides), in treating diseases and disorder ofthe skeletal system (e.g., osteoporosis and osteoarthritis), in treatingdiseases and disorders of the cardiovascular system (e.g., stroke, heartdisease, atherosclerosis, restenosis, thrombosis, anemia, leucopenia,neutropenia, thrombocytopenia, granuloctopenia, pancytoia or idiopathicthrombocytopenic purpura); in treating diseases and disorders of thekidneys (e.g., nephropathy), pancreas (e.g., pancreatitis), skin andeyes (e.g., conjunctivitis, retinitis, scleritis, uveitis, allergicconjuctivitis, vernal conjunctivitis, pappillary conjunctivitisglaucoma, retinopathy, and ocular ischemic conditions including anteriorischemic optic neuropathy, age-related macular degeneration (AMD),Ischemic Optic Neuropathy (ION), dry eye syndrome); in preventing organtransplantation rejection (e.g., lung, liver, heart, pancreas, andkidney transplantation) and uses in regenerative medicine (e.g., incounteracting aging, in promoting wound healing and stimulating bone,collagen, tissue and organ growth and repair).

In various embodiments, the invention provides compositions for use inmodulating a target nucleic acid or protein in a cell, in vivo in asubject, or ex vivo. The HES-oligonucleotide compostions of theinvention have applications in for example, treating a disease ordisorder characterized by an overexpression, underexpression and/oraberrant expression of a nucleic acid or protein in a subject in vivo orex vivo. Uses of the compositions of the invention in treating exemplarydiseases or disorders selected from: an infectious disease, cancer, aproliferative disease or disorder, a neurological disease or disorder,and inflammatory disease or disorder, a disease or disorder of theimmune system, a disease or disorder of the cardiovascular system, ametabolic disease or disorder, a disease or disorder of the skeletalsystem, and a disease or disorder of the skin or eyes are alsoencompassed by the invention.

As one of skill in the art will immediately appreciate, the therapeuticand companion diagnostic uses of the HES-oligonucleotides of theinvention are essentially limitless. Provided herein are exemplarydiagnostic and therapeutic uses of the compositions of theHES-oligonucleotides of the invention. However, the description hereinis not meant to be limiting and it is envisioned that theHES-oligonucleotides have uses in any situations where it is desirableto detect a nucleic acid sequence or to modulate levels of one or morenucleic acids or related proteins in a cell and/or organism.

Plurality of HES-Oligonucleotides

In some embodiments, the pharmaceutical compositions of the inventioncomprise a combination of at least 2, at least 3, at least 4, at least5, or at least 10 different HES-oligonucleotide complexes havingdifferent oligonucleotide sequences. In some embodiments, thepharmaceutical compositions contain between 2-15, 2-10, or 2-5 differentHES-oligonucleotide complexes. In some embodiments, at least 2 or atleast 3 of the different oligonucleotides in the complex specificallyhybridize to a DNA and/or mRNA corresponding to the same polypeptide. Insome embodiments, at least 2, at least 3, at least 4, at least 5, or atleast 10 of the different oligonucleotides in the complex specificallyhybridizes to a DNA and/or mRNA corresponding to different polypeptides.In some embodiments, the pharmaceutical compositions contain between2-15, 2-10, or 2-5 oligonucleotides that specifically hybridize todifferent polypeptides. In some embodiments, one or more of thedifferent HES-oligonucleotides are administered to a subjectconcurrently. In other embodiments, one or more of the differentHES-oligonucleotides are administered to a subject separately.

In certain embodiments, an HES-oligonucleotide complex of the inventionis co-administered with one or more additional agents. In certainembodiments, such additional agents are designed to treat a differentdisease, disorder, or condition as the HES-oligonucleotide complex. Insome embodiments, the additional agent is co-administered with theHES-oligonucleotide complex to treat an undesired effect of the complex.In additional embodiments, the additional agent is co-administered withthe HES-oligonucleotide complex to produce a combinational effect. Infurther embodiments, the additional agent is co-administered with theHES-oligonucleotide complex to produce a synergistic effect. In certainembodiments, the additional agent is administered to treat an undesiredside effect of an HES-oligonucleotide complex of the invention. In someembodiments, the HES-oligonucleotide complex is administered at the sametime as the additional agent. In some embodiments, theHES-oligonucleotide and additional agent are prepared together in asingle pharmaceutical formulation. In other embodiments, theHES-oligonucleotide and additional agent are prepared separately. Infurther embodiments, the additional agent is administered at a differenttime from the HES-oligonucleotide complex.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth. In addition, the term ‘cell’can be construed as a cell population, which can be either heterogeneousor homogeneous in nature, and can also refer to an aggregate of cells.Moreover, each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, envisioned that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each embodiment of theinvention.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose of skill in the art, may be made without departing from the spiritand scope of the present invention.

The disclosure of U.S. Appl. No. 61/630,446 is herein incorporated byreference in its entirety. Moreover, all publications, patents, patentapplications, internet sites, and accession numbers/database sequences(including both polynucleotide and polypeptide sequences) cited areherein incorporated by reference in their entirety for all purposes tothe same extent as if each individual publication, patent, patentapplication, internet site, or accession number/database sequence werespecifically and individually indicated to be so incorporated byreference. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents are based on the information availableto the applicants and do not constitute any admission as to thecorrectness of the dates or contents of these documents.

EXAMPLES

The following examples which are offered to illustrate, but not tolimit, the claimed invention, clearly show: (1) the presence of an HESallows delivery of oligonucleotides inside live cells without toxicityin a living organism (2) the formation of an HES in a double-strandedRNA (3) the absence of inhibition by an HES of processing of adouble-stranded RNA (dsRNA) by the endonuclease Dicer and (4) theknockdown of a gene by a dsRNA containing an H-type excitonic structure.

Example 1 In Vivo Delivery of an Oligonucleotide Containing an H-TypeExcitonic Structure

In order to show that oligonucleotides can be delivered inside livecells without toxicity in a live organism, a strand of DNA containing asequence of 24 nucleic acids complementary to β-actin (CCC GGC GAT ATCATC ATC CAT AAC (SEQ ID NO:1) (Sokol et al. Proc. Natl. Acad. Sci. USA95:11538-43 (1998)) was synthesized and covalently labeled on oppositeends of the strand with the fluorophore(N-Ethyl-N′-[5-(N″-succinimidyloxycarbonyl)pentyl]-3,3,3′,3′,-tetramethyl-2,2′-indodicarbocyaninechloride). The labeled oligonucleotide was purified by reverse phasehigh pressure liquid chromatography (hplc) and then lyophilized. Thepresence of an intramolecular HES in the oligonucleotide was confirmedby absorbance spectrometry and fluorometry. All measurements werecarried out in phosphate buffered saline (PBS) in which the labeledoligonucleotide was readily solubilized.

A volume of two hundred microliters of the labeled oligonucleotide at aconcentration of 5 micromolar in PBS was injected into the tail vein ofa six week old C57BL/6 mouse (464 micrograms/kilogram). After 18 hours,the mouse was sacrificed by cervical dislocation; blood was immediatelywithdrawn from the heart and the spleen was removed. The blood wasdiluted with PBS, placed over Hypaque-Ficoll, and centrifuged at 1300rpm for 30 minutes. Cells at the interface between the Hypaque-Ficolland PBS were collected, washed with PBS, placed on a #0 borosilicateglass surface in a Mattek glass bottom microwell dish (P35G-0-10-C),allowed to settle (ca. 10 minutes), and then imaged with a Leica DMIRE2confocal microscope. In parallel a single cell suspension from thespleen was made by applying the end of a syringe to the resected organand then triturating the suspension. The splenocytes in PBS were thenexposed to an equal volume of ACK lysis buffer for 3 minutes, dilutedfurther with PBS, and centrifuged. Cells in the pellet were thenresuspended in PBS, placed in a Mattek glass bottom microwell dish(P35G-0-10-C), allowed to settle (ca. 10 minutes), and finally imagedwith a confocal microscope.

Imaging of the blood and splenocyte samples was carried out by acquiringa series of stacks of 1 micron sections in both the fluorescence andbrightfield (differential interference contrast (DIC)) channels. Imageswere reconstructed by overlaying the sections of each channel to producea condensed stack then overlaying the condensed images from fluorescenceand DIC channels.

Images showed the fluorescence channels overlayed on the DIC imagesindicated all splenocytes and blood cells took up the HES-containingoligonucleotide. The presence of oligonucleotide inside live cells wasconfirmed by examination of each 1 micron section. As was also evident,particularly from the DIC images, cells from both blood and spleen werehealthy, a point further substantiated by the lack of uptake of trypanblue or propidium iodide by cells in these same samples.

Example 2 Quantitation of In Vivo Delivery of an OligonucleotideContaining an H-Type Excitonic Structure

In order to quantitate the in vivo delivery of oligonucleotides insidelive cells without toxicity in a live organism, a Dicer substrate wasprepared as described in Example 3. The sequence for the Dicersubstrate, i.e., the sense strand and antisense strand for eGFP (Kim etal. Nature Biotech. 22:321-5 (2004)), was chosen so that nocomplementary pairing in the subject mice (standard, nontransfectedBALB/C strain) could take place. The double-labeled lyophilized dsRNAwas solubilized in phosphate buffered saline (PBS). The presence of anintramolecular HES in the oligonucleotide was confirmed by absorbancespectrometry and fluorometry.

A volume of two hundred microliters of the labeled oligonucleotide at aconcentration of 5 micromolar or 10 micromolar in PBS was injected intothe tail vein or the peritoneum of each 10-12 week old BALB/C mouse(0.75 or 1.5 milligrams/kilogram). After 3 hours, blood was drawn in thepresence of heparin from the heart of each mouse. The blood was dilutedwith PBS, placed over Hypaque-Ficoll, and centrifuged at 1300 rpm for 30minutes. Cells at the interface between the Hypaque-Ficoll and PBS werecollected; the fluorescence of individual cells was measured with aCytek-modified Becton-Dickinson Caliber flow cytometer,

FIG. 1 shows histograms of blood cells isolated from mice three hoursafter an injection of 200 microliters of buffer (PBS) or a Dicersubstrate. In Panel a, fluorescence from cells which were isolated aftera single ip injection of PBS or the Dicer substrate (1.5 mg/kg) is shownin histogram format. The increase in fluorescence intensity of ca. 2logs in the cells exposed to the Dicer substrate relative to those fromthe animal that had received an injection of PBS indicates significantuptake of the Dicer substrate. Moreover, the light scattering propertiesof both groups indicated highly viable cells. In Panel b, histogramsshow the fluorescence of cells isolated after an iv injection of PBS,the Dicer substrate at a concentration of 1.5 mg/kg, or the Dicersubstrate at a concentration of 0.75 mg/kg. As with the ip route, cellsfrom iv-injected animals that had received the Dicer substrate at eitherdose also showed ca. a two log increase in fluorescence intensity percell relative to those from the PBS animal with the higher concentrationresulting in a slightly higher average intensity per cell. And, again,no signs of toxicity were observed.

Example 3 Formation of an Intramolecular HES in Real-Time

The formation of an HES is associated with quenching of fluorescence;

specifically, the fluorescence of the dimer is reduced relative to thatof the individual components. Therefore, in order to illustrate theprocess of HES formation, two complementary strands of RNA, i.e., thesense strand and antisense strand (Kim et al. Nature Biotech. 22:321-5(2004)), were each labeled withN-Ethyl-N′-[5-(N″-succinimidyloxycarbonyl)pentyl]-3,3,3′,3′,-tetramethyl-2,2′-indodicarbocyaninechloride and then added together; the fluorescence intensity of thelatter solution was then compared with those of the components, i.e.,the single strands alone.

The fluorescence spectra of the two singly-labeled strands are shown inthe top two panels on the left side of FIG. 2. The purity of each strandas measured by reverse phase hplc is also shown in the correspondingpanels on the right side of FIG. 2.

With a data acquisition rate of 1 datum/sec. the center section shows,first, the fluorescence intensity of the sense solution as a function oftime (from 0 to ca. 80 sec.) to be ca. 7000 Counts. When the shutter isclosed at 80 sec. in order to add the antisense solution, the intensitydrops to the zero. Upon re-opening the shutter, the intensity isrecorded at ca. 1100 Counts and remains steady at this level due to thetight complex formed between the sense and antisense strands.

The lowest panels on the right and left sides show the emission spectrumand hplc chromatogram of the sense-antisense complex, respectively.

Example 4 Recognition of the a Double-Stranded Sense-Antisense RNAComplex by Dicer

Dicer is an endonuclease that cleaves double-stranded RNA (dsRNA) andpreMiRNA (MiRNA) into short double-stranded RNA fragments called smallinterfering siRNA. Since one of the embodiments of this invention is thedelivery of oligonucleotides for silencing RNA, it is essential that anHES-containing dsRNA be recognizable and cleavable by Dicer. Therefore,the dsRNA described in Example 3 which contains an HES on the end of theduplex was exposed to a recombinant Dicer (Recombinant Turbo Dicer Cat(# T520002) from Genlantis). Using the digestion conditions in theinstructions from the reagent supplier the fluorescence of thedsRNA-containing solution was measured after addition of thisendonuclease.

Two Dicer substrates derivatized with an HES were synthesized: one wascomprised of two strands of unmodified ribonucleotides (25 and 27 bases)and a second with the same two strands but with the 25 nucleotide chainextended with two O-methylated nucleotides on the end. TerminalO-methylation has been shown to protect oligonucleotides fromexonucleases present in plasma. As shown in FIG. 3, the fluorescence ofthe solutions of both dsRNAs increased as a function of time afteraddition of Dicer, thus confirming the absence of inhibition of the HESfor processing by this endonuclease. Additionally, the dsRNA with theO-methylation showed a slightly slower rate of digestion, consistentwith the protective effect of this modification.

Example 5 Knockdown of a Gene by a dsRNA Containing an H-Type ExcitonicStructure

In order to show the functionality of an oligonucleotide linked to anH-type excitonic structure, the fluorescence per cell from blood cellsof mice transgenic for expression of eGFP was measured after exposure toa double-stranded RNA (dsRNA) derivatized with an H-type excitonicstructure, as described in FIG. 2, and containing the sense andantisense strands coding for eGFP (Kim et al, Nature Biotech. 22:321-5(2004)). Measurements were made by flow cytometry from the blood of miceafter separation of mononuclear cells.

FIG. 4 shows the superimposed histograms of both the control andDicer-treated populations. The control cells show two populations: ca.67% of cells with >10² fluorescence units per cell than a secondnonfluorescent population. Treatment with the Dicer substrate results ina single population with an average fluorescence just slightly abovethat of the nonfluorescent control cells.

What is claimed is:
 1. A method for modulating a target nucleic acid orprotein in a subject, said method comprising administering to thesubject an effective amount of an H-type excitonic structure(HES)-oligonucleotide containing a therapeutic oligonucleotide thatspecifically hybridizes to a target nucleic acid sequence in vivo andthereby modulates the level of a protein encoded or regulated by thenucleic acid.
 2. A method for treating a disease or disorder in asubject, said method comprising administering to a subject in needthereof, an effective amount of an H-type excitonic structure(HES)-oligonucleotide containing a therapeutic oligonucleotide thatspecifically hybridizes to a nucleic acid sequence in vivo and modulatesthe level of a protein encoded or regulated by the nucleic acid; whereinthe disease or disorder is characterized by (a) overexpression orunderexpression of the target a nucleic acid in the subject, or (b)overexpression or underexpression of a protein encoded by the targetnucleic in the subject.
 3. The method of claim 1, wherein thetherapeutic oligonucleotide can induce RNA interference (RNAi).
 4. Themethod of claim 1, wherein the therapeutic oligonucleotide is a memberselected from: siRNA, shRNA, miRNA, a Dicer substrate, and an antisense.5. The method of claim 1, wherein the therapeutic oligonucleotide is aDicer substrate.
 6. The method of claim 5, wherein the Dicer substratecomprises a nucleic acid strand 18-35 nucleotides in length.
 7. Themethod of claim 5, wherein the Dicer substrate comprises a nucleic acidstrand 18-25 nucleotides in length.
 8. The method of claim 5, whereinthe Dicer substrate comprises 2 complementary nucleic acid strands thatare each 18-35 nucleotides in length and wherein the complementarynucleic acid strands form a 2 nucleotide 3′ overhang.
 9. The method ofclaim 5, wherein the Dicer substrate comprises 2 complementary nucleicacid strands that are each 18-25 nucleotides in length and wherein thecomplementary nucleic acid strands form a 2 nucleotide 3′ overhang. 10.The method of claim 1, wherein the therapeutic oligonucleotide containsone or more modified nucleoside motifs selected from: locked nucleic add(LNA), alpha LNA, 2′-Fluoro (2′F), 2′-O(CH₂)₂OCH₃(2′-MOE),2′-OCH₃(2′-O-methyl) (2′OME), PNA, and morpholino.
 11. The method ofclaim 1, wherein the therapeutic oligonucleotide contains one or moremodifications selected from: phosphorothioate, phosphorodithioate,phosphoramide, 3′-methylene phosphonate, O-methylphosphoroamidiate, PNA,morpholino, C-5 propyne, and 5-methyl C.
 12. The method of claim 2,wherein the therapeutic oligonucleotide can induce RNA interference(RNAi).
 13. The method of claim 2, wherein the therapeuticoligonucleotide is a member selected from: siRNA, shRNA, miRNA, a Dicersubstrate, and an antisense.
 14. The method of claim 2, wherein thetherapeutic oligonucleotide is a Dicer substrate.
 15. The method ofclaim 14, wherein the Dicer substrate comprises a nucleic acid strand18-35 nucleotides in length.
 16. The method of claim 14, wherein theDicer substrate comprises a nucleic acid strand 18-25 nucleotides inlength.
 17. The method of claim 14, wherein the Dicer substratecomprises 2 complementary nucleic acid strands that are each 18-35nucleotides in length and wherein the complementary nucleic acid strandsform a 2 nucleotide 3′ overhang.
 18. The method of claim 14, wherein theDicer substrate comprises 2 complementary nucleic add strands that areeach 18-25 nucleotides in length and wherein the complimentary nucleicacid strands form a 2 nucleotide 3′ overhang.
 19. The method of claim10, wherein the therapeutic oligonucleotide contains one or moremodified nucleoside motifs selected from: locked nucleic add (LNA),alpha LNA, 2-Fluoro (2′F), 2′-O(CH₂)₂OCH₃(2′-MOE), 2′-OCH₃(2′-O-methyl)(2′OME), PNA and morpholino.
 20. The method of claim 2, wherein thetherapeutic oligonucleotide contains one or more modifications selectedfrom: phosphorothioate, phosphorodithioate, phosphoramide, 3′-methylenephosphonate, O-methylphosphor-oamidiate, PNA, morpholino, C-5 propyne,and 5-methyl C.
 21. The method of claim 1, wherein the therapeuticoligonucleotide contains 1 or 2 substitutions, deletions or insertionscompared to the corresponding reverse complementary strand of a targetRNA.